CLDN18.2 / 4-1BB binding protein and medical application thereof

Abstract

The invention relates to a CLDN18.2 / 4-1BB binding protein and a medical application thereof. In particular, the present disclosure relates to CLDN18.2 / 4-1BB binding proteins, 4-1BB binding proteins, CD16A binding proteins, methods of using the same for the treatment of cancer, and related pharmaceutical uses of the CLDN18.2 / 4-1BB binding proteins, 4-1BB binding proteins, CD16A binding proteins.

Description

CLDN18.2 / 4-1BB binding protein and its medical use

[0001] This application claims priority to Chinese patent application CN202211513673.8 filed on November 29, 2022, and the entire contents of the above patent application are incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of biomedicine, and specifically to CLDN18.2 / 4-1BB binding protein, 4-1BB binding protein, CD16A binding protein, methods for treating cancer, and related pharmaceutical uses thereof. Background Art

[0003] The claudin protein family is an important class of tight junction proteins between cells, generally composed of four transmembrane domains and two extracellular loops. Their primary function is to maintain normal molecular exchange between cells. Claudin18, a member of the claudin protein family, has two splice variants: Claudin18 splice variant 1 (CLDN18.1) and 2 (CLDN18.2). The two variants differ only by eight amino acids in the first extracellular segment. The two variants have distinct physiological distribution differences: CLDN18.1 is concentrated in normal lung tissue, while CLDN18.2 is primarily expressed in the stomach. CLDN18.2 in healthy tissue is typically buried in the gastric mucosa, making it largely inaccessible to antibodies. However, when cells undergo malignant transformation, intercellular junctions are disrupted, exposing CLDN18.2 to antibody-binding epitopes, making it a tumor-specific target. CLDN18.2 is significantly ectopically expressed in a variety of epithelial tumors, including gastric, esophageal, and pancreatic cancers, providing a biological basis for targeted tumor therapy targeting CLDN18.2. IMAB362, an IgG1 anti-CLDN18.2 antibody developed by Astellas, achieved a 63.2% response rate in combination with chemotherapy in patients with advanced metastatic gastric and gastroesophageal cancer, surpassing historical data from chemotherapy alone (2021 ASCO). This demonstrates the clinical value of CLDN18.2 as a tumor-specific antigen.

[0004] 4-1BB (CD137, TNFRSF9) is a transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. + and CD4 +A co-stimulatory molecule on the surface of cells such as T cells, regulatory T cells (Tregs), NK cells and NKT cells, B cells and neutrophils. On T cells, 4-1BB is not constitutively expressed, but is induced to express after activation of the T cell receptor (TCR). 4-1BB is expressed on the cell surface in the form of monomers or dimers. After binding to its natural ligand 4-1BBL, it forms a trimer and transmits signals through TNFR-associated factor (TRAF)-2 and TRAF-1. The early signaling of 4-1BB involves the polyubiquitination of K-63, which activates the nuclear factor (NF-κB) and mitogen-activated protein kinase (MAPK) pathway signaling pathways, leading to the proliferation, maturation and survival of T cells, as well as the production of cytokines. Studies have shown that antibody agonists targeting 4-1BB can promote the anti-tumor function of T cells in mice (Murillo et al., Clin Cancer Res. 2008; 14(21): 6895-906). Antibodies that activate 4-1BB can increase the expression of co-stimulatory molecules in many models, induce T cell survival and proliferation, thereby enhancing anti-tumor immune responses and causing immune cells to kill tumors. Existing 4-1BB-activating antibodies include Bristol Myers Squibb (BMS)'s Urelumab, a human IgG4 antibody (WO2005035584); Pfizer's Utomilumab, a human IgG2 antibody (Fisher et al., Cancer Immunol. 2012; 61: 1721-1733); and Adagene's ADG106, a human IgG4 antibody (WO2019037711A1). However, the development of anti-tumor antibodies targeting 4-1BB has not been smooth, mainly because 4-1BB can activate T cells in the periphery (e.g., the liver) in addition to activating T cells within the tumor, triggering liver inflammation and leading to severe liver damage (Todd Bartkowiak et al. Clin Cancer Res; 24(5) March 1, 2018). This narrows the therapeutic window of 4-1BB.

[0005] In view of the excellent tumor specificity and targeting of CLDN18.2, and the problems with the agonistic antibodies targeting 4-1BB in the prior art, the present disclosure provides an anti-4-1BB single domain antibody of a new structure, designs and develops an anti-CLDN18.2 / 4-1BB bispecific antibody, which relies on CLDN18.2 on tumor tissue to activate 4-1BB, can avoid the hepatotoxicity caused by 4-1BB, and expands the therapeutic window of the antibody. When an enhanced Fc of effector function (such as ADCC, ADCP) is used in the anti-CLDN18.2 / 4-1BB bispecific antibody, the effect is further improved. In addition, the present disclosure also provides an anti-CD16A single domain antibody of a new structure, which forms an anti-CLDN18.2 / 4-1BB / CD16A trispecific antibody together with an anti-CLDN18.2 antibody and an anti-4-1BB single domain antibody, wherein the anti-CD16A single domain antibody can enhance the ADCC function of the antibody. The anti-CLDN18.2 / 4-1BB bispecific antibody and the anti-CLDN18.2 / 4-1BB / CD16A trispecific antibody disclosed herein can prevent the antibody from killing 4-1BB-positive T cells through ADCC, which helps to improve the anti-tumor effect and has good safety and effectiveness. At the same time, the antibodies disclosed herein have good pharmacological activity, drugability, and expression level. In summary, the antibodies disclosed herein have the potential to become excellent candidate drugs for the clinical treatment of tumors.

[0006] Summary of the Invention

[0007] The present disclosure provides 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, CLDN18.2 / 4-1BB / CD16A binding protein, their encoding nucleic acids, vectors, host cells, pharmaceutical compositions, methods for treating or preventing cancer, and related pharmaceutical uses.

[0008] 4-1BB binding protein

[0009] The present disclosure provides a 4-1BB binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 10, 18-21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. The immunoglobulin single variable structure specifically binds to the 4-1BB antigen or a fragment thereof.

[0010] In some embodiments, a 4-1BB binding protein is provided, comprising any one or any combination of the above-mentioned CDR1, CDR2, and CDR3.

[0011] In some embodiments, a 4-1BB binding protein is provided, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 11, 12, and 13, respectively, or in SEQ ID NOs: 11, 12, and 22. These are CDRs defined according to the Kabat numbering system.

[0012] In some embodiments, the immunoglobulin single variable domain in the aforementioned 4-1BB binding protein is humanized, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0013] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing TCE, and has one or more changes in one or more CDRs, which result in reduced immunogenicity of the 4-1BB binding protein.

[0014] In some embodiments, the immunoglobulin single variable domain is humanized. The heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-64*04, IGHV3-23*03, and / or IGHV3-74*01. In some embodiments, FR1 is derived from IGHV3-64*04, FR2 is derived from IGHV3-23*03, and FR3 is derived from IGHV3-74*01.

[0015] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned 4-1BB binding protein is shown as any one of SEQ ID NOs: 10, 18-21, or has at least 80% or at least 90% sequence identity with any one of SEQ ID NOs: 10, 18-21.

[0016] In the present disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0017] In some embodiments, the aforementioned 4-1BB binding protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or a fragment thereof.

[0018] In some specific embodiments, the antibody or antigen-binding fragment thereof is a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv).

[0019] In some embodiments, the immunoglobulin single variable domain in the aforementioned 4-1BB binding protein is a single domain antibody or VHH.

[0020] In some embodiments, the present disclosure provides a 4-1BB binding protein comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, wherein the immunoglobulin single variable domains may be the same or different and may form dimers or multimers.

[0021] In some embodiments, the aforementioned 4-1BB binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, or IgG4.

[0022] In some embodiments, the Fc has a mutation at position C220, such as C220A.

[0023] In some embodiments, the Fc region is an Fc region that increases effector function, e.g., increases antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) with increased effector function.

[0024] Exemplary IgG1 Fc regions include those with the following substitutions: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 280H / 290S / 298V;243L / 292P / 300L;243L / 292P / 300L / 396L;243L / 292P / 300L / 305I / 396L;236A / 239D / 332E;326A / 333A;326W / 333S;290E / 298G / 299A;290N / 298G / 299A;290E / 298G / 299A / 326E;or 290N / 298G / 299A / 326E;or any combination of any of the above positions. Said mutations are defined according to the EU numbering system.

[0025] Exemplary IgG1 Fc regions include those having the following substitutions: S239D; S239E; S239K, F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A3 30L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D280H / K290S / S298V; F243 L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; or K290N / S298G / T299A / K326E, or any combination of any of the foregoing positions.

[0026] In some embodiments, the Fc region is an Fc region with reduced effector function, eg, reduced ADCC, ADCP, and / or CDC. Exemplary Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0027] In some specific embodiments, the aforementioned 4-1BB binding protein comprises an Fc region of human IgG1, and the Fc has C220A / S267E / L328F, C220A / L234A / L235A / N297A, S267E / L328F or L234A / L235A / N297A mutations. In some specific embodiments, the aforementioned 4-1BB binding protein comprises an Fc region of human IgG4, and the Fc region of human IgG4 has an S228P mutation.

[0028] In some embodiments, the aforementioned 4-1BB binding protein further comprises a human immunoglobulin Fc region, wherein the Fc region is as shown in any one of SEQ ID NOs: 14-16, or has at least 80% or at least 90% sequence identity with any one of SEQ ID NOs: 14-16.

[0029] In some embodiments, a 4-1BB binding protein is provided, which has an amino acid sequence as shown in SEQ ID NO: 17 or has at least 80% or at least 90% sequence identity thereto.

[0030] In the context of mutations contained in the Fc region in the present disclosure, " / " means "and", for example, "L234A / L235A" means "L234A and L235A", that is, the Fc contains L234A and L235A mutations; the amino acid positions of the mutations in the Fc region of the present disclosure are all defined according to the EU numbering system.

[0031] In some embodiments, the Fc region contained in the aforementioned 4-1BB binding protein can enable the binding protein to form a dimeric molecule.

[0032] In some embodiments, the Fc region contained in the aforementioned 4-1BB binding protein can extend the in vivo half-life of the binding protein.

[0033] In some embodiments, the immunoglobulin single variable domain in the aforementioned 4-1BB binding protein is connected to the Fc region directly or through a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structure. For example, the linker is a flexible linker, such as G4S (SEQ ID NO: 102), GS, GAP, (G4S) 2 (SEQ ID NO: 103), (G4S) 3 (SEQ ID NO: 104), (G4S) 4 (SEQ ID NO: 105), (G4S) 5 (SEQ ID NO: 106), ASGS (SEQ ID NO: 107), etc., and more preferably (G4S) 2.

[0034] In some embodiments, the 4-1BB binding protein of the present disclosure is an anti-4-1BB antibody or an antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.

[0035] In some embodiments, the aforementioned 4-1BB binding protein has at least one activity selected from the following:

[0036] (a)≤10 -7 K D The value binds to human 4-1BB or its epitope;

[0037] (b) weak or no activation of the 4-1BB signaling pathway when not cross-linked with FcγRIIb (i.e., CD32b), e.g., at 100 nM antibody concentration, the degree of activation without FcγRIIb cross-linking is no more than 10% of the activity at saturating antibody concentration when cross-linked with FcγRIIb;

[0038] (c) When cross-linked by FcγRIIb, the 4-1BB signaling pathway is strongly activated or strongly activated, e.g., EC 50 Less than 1nM;

[0039] (d) activating T cells and / or promoting T cell proliferation;

[0040] (e) inhibiting tumor growth;

[0041] For example, the detection of activation of the 4-1BB signaling pathway in (b) and (c) can be found in the 4-1BB / NF-κB luciferase reporter gene detection method of Example 2.

[0042] In some embodiments, the aforementioned 4-1BB binding protein of the present disclosure binds to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9M, or ≤1×10 -10 M.

[0043] In some embodiments, the aforementioned 4-1BB binding protein of the present disclosure can inhibit tumor growth by at least about 10%, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0044] In some embodiments, the aforementioned 4-1BB-binding protein disclosed herein encompasses variants, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 10, 18-21; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or the FR region.

[0045] In some embodiments, an anti-4-1BB antibody or an antigen-binding fragment thereof is provided, which binds to or competes for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned 4-1BB-binding protein of the present disclosure.

[0046] In some embodiments, an anti-4-1BB antibody or an antigen-binding fragment thereof is provided, which blocks the binding of the immunoglobulin single variable domain to 4-1BB (e.g., human 4-1BB) in the aforementioned 4-1BB binding protein of the present disclosure. In some specific embodiments, the anti-4-1BB antibody or its antigen-binding fragment can also activate T cells and / or promote T cell proliferation.

[0047] In some embodiments, an anti-4-1BB antibody or antigen-binding fragment thereof is provided, wherein the binding of the anti-4-1BB antibody to 4-1BB (eg, human 4-1BB) is blocked by the immunoglobulin single variable domain in the aforementioned 4-1BB binding protein of the present disclosure.

[0048] In some embodiments, there is provided a protein or molecule comprising any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) immunoglobulin single variable domains in the aforementioned disclosure, the immunoglobulin single variable domains being the same or different. For example, the protein or molecule is a conjugate, and the conjugate may, for example, include any detectable label.

[0049] CD16A binding protein

[0050] The present disclosure provides a CD16A binding protein comprising an immunoglobulin single variable domain comprising:

[0051] CDR1, CDR2, and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 23, 35-39, or CDR1, CDR2, and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 24, 40-43;

[0052] Wherein, the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0053] In some embodiments, a CD16A binding protein is provided, which comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises any one or any combination of the above-mentioned CDR1, CDR2 and CDR3.

[0054] In some embodiments, a CD16A binding protein is provided, comprising an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3, wherein:

[0055] The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 25, 26 and 27, respectively.

[0056] The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 28, 29 and 30, respectively.

[0057] Alternatively, the amino acid sequences of the CDR1, CDR2 and CDR3 are shown as SEQ ID NOs: 28, 44 and 30, respectively; wherein the CDR1, CDR2 and CDR3 are CDRs defined according to the Kabat numbering system.

[0058] In some embodiments, the immunoglobulin single variable domain in the aforementioned CD16A binding protein is humanized, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced and / or antibody isomerization reduced.

[0059] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing TCE, and has one or more changes in one or more CDRs, which result in reduced immunogenicity of the CD16A binding protein.

[0060] In some embodiments, the immunoglobulin single variable domain is humanized. The heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-23*04 or IGHV3-20*04. In some embodiments, when the parent immunoglobulin single variable domain is SEQ ID NO: 23, the heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-23*04; when the parent immunoglobulin single variable domain is SEQ ID NO: 24, the heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-20*04.

[0061] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned CD16A binding protein is as shown in any one of SEQ ID NOs: 23, 35-39, or has at least 80% or at least 90% sequence identity with any one of SEQ ID NOs: 23, 35-39; or, as shown in any one of SEQ ID NOs: 24, 40-43, or has at least 80% or at least 90% sequence identity with any one of SEQ ID NOs: 24, 40-43.

[0062] In some embodiments, the immunoglobulin single variable structure in the aforementioned CD16A binding protein specifically binds to CD16A but does not specifically bind to CD16B.

[0063] In some embodiments, the aforementioned CD16A binding protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to CD16A. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody, and tetrabody, tandem two-scFv, tandem three-scFv).

[0064] In some embodiments, the immunoglobulin single variable domain in the aforementioned CD16A binding protein is a single domain antibody or VHH.

[0065] In some embodiments, the present disclosure provides a CD16A binding protein comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, which may be the same or different and may form dimers or multimers.

[0066] In some embodiments, the aforementioned CD16A binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, or IgG4. In some embodiments, the Fc region can be an Fc region with enhanced effector function, wherein the effector function is ADCC, ADCP, and / or CDC. For example, the Fc region can have mutations, and exemplary IgG1 Fc regions with increased effector function include those having the following substitutions or any combination thereof: S239D, S239E, S239K, F241A, V262A, V264D, V264L, V264A, V264S, D265A, D265S, D265V, F296A, Y296A, R301A, I332E, S239D / I332E, S239D / A330S / I332E, S239D / A330L / I332E, S298A / D333A / K334A, P247I / A339D, P247I / A339Q, D280H / K290S, 396L, K290E / S298G / T299A, K290N / S298G / T299A, K290E / S298G / T299A / K326E, or K290N / S298G / T299A / K326E, or any combination of the foregoing positions.

[0067] In other embodiments, the Fc region can be an Fc region with reduced effector function, for example, the Fc region can have a mutation, and exemplary IgG Fc regions with reduced effector function include those having the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0068] The mutations used in this disclosure that can increase or decrease the effector function of the Fc region are well known in the art, such as those disclosed in WO2019220369A, WO2021027850A, WO2020180712A, etc., and are incorporated herein by reference in their entirety.

[0069] In some specific embodiments, the Fc region is as shown in any one of SEQ ID NOs: 14-16, or has at least 80% or at least 90% sequence identity to any one of SEQ ID NOs: 14-16.

[0070] In some embodiments, the Fc region contained in the aforementioned CD16A binding protein can enable the binding protein to form a dimeric molecule and prolong the in vivo half-life of the binding protein.

[0071] In some embodiments, the immunoglobulin single variable domain in the aforementioned CD16A binding protein is connected to the Fc region directly or through a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structure. For example, the linker is a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, etc., and more preferably (G4S)2.

[0072] In some embodiments, the CD16A binding protein of the present disclosure is an anti-CD16A antibody or an antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.

[0073] In some embodiments, the aforementioned CD16A binding protein has at least one activity selected from the group consisting of:

[0074] (a)≤10 -7 K D The value specifically binds to human CD16A or its epitope;

[0075] (b) no binding or weak binding to human CD16B or its epitope-specific binding, wherein the weak binding is 10% or less of the affinity for human CD16A or its epitope-specific binding;

[0076] (c) binds to the CD16A 158V (i.e., CD16A 176V) variant and CD16A 158F (i.e., CD16A 176F) with the same or similar affinity;

[0077] (d) activating or enhancing ADCC via NK cells;

[0078] (e) activating or enhancing ADCP via macrophages;

[0079] (f) Inhibit tumor growth.

[0080] In some embodiments, the aforementioned CD16A binding protein of the present disclosure binds to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0081] In some embodiments, the aforementioned CD16A binding proteins of the present disclosure are capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0082] In some embodiments, the aforementioned CD16A binding proteins disclosed herein encompass variants, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 23-24, 35-43; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or the FR region.

[0083] In some embodiments, an anti-CD16A antibody or antigen-binding fragment thereof is provided, which binds to or competes for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned CD16A binding protein of the present disclosure.

[0084] In some embodiments, an anti-CD16A antibody or an antigen-binding fragment thereof is provided, which blocks the binding of the immunoglobulin single variable domain in the aforementioned CD16A binding protein of the present disclosure to CD16A (eg, human CD16A).

[0085] In some embodiments, an anti-CD16A antibody or antigen-binding fragment thereof is provided, wherein the binding of the anti-CD16A antibody or antigen-binding fragment thereof to CD16A (eg, human CD16A) is blocked by the immunoglobulin single variable domain in the aforementioned CD16A binding protein of the present disclosure.

[0086] In some embodiments, a protein or molecule is provided that comprises an immunoglobulin single variable domain from any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) CD16A binding proteins disclosed herein, wherein the immunoglobulin single variable domains are the same or different. For example, the protein or molecule is a conjugate, which may, for example, comprise any detectable label.

[0087] CLDN18.2 / 4-1BB binding protein

[0088] In a first aspect, the present disclosure provides a CLDN18.2 / 4-1BB binding protein, which comprises a first antigen-binding domain that specifically binds to 4-1BB and a second antigen-binding domain that specifically binds to CLDN18.2, and can specifically bind to 4-1BB and CLDN18.2 simultaneously or separately.

[0089] In some embodiments, when the second antigen-binding domain that specifically binds to CLDN18.2 does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB does not activate 4-1BB signaling, or binds to the 4-1BB antigen but does not activate 4-1BB signaling.

[0090] In some embodiments, the CLDN18.2 / 4-1BB binding protein has enhanced effector function, for example, the CLDN18.2 / 4-1BB binding protein comprises an Fc region with enhanced effector function and / or a third antigen binding domain that specifically binds to CD16A.

[0091] In some embodiments, the Fc region with enhanced effector function increases binding to FcγR, for example, increases binding to FcγRIIB (CD32B) or increases binding to FcγRIIIA (CD16A). In some embodiments, the binding to FcγRIIIB (CD16B) is reduced, decreased, or absent.

[0092] In some embodiments, the Fc region with enhanced effector function is enhanced binding to C1q.

[0093] In some embodiments, the Fc region with enhanced effector function has a normal or higher glycosylation level compared to a wild-type Fc region.

[0094] In some embodiments, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0095] In a second aspect, the present disclosure provides a CLDN18.2 / 4-1BB binding protein, which comprises a first antigen-binding domain that specifically binds to 4-1BB, a second antigen-binding domain that specifically binds to CLDN18.2, and a third antigen-binding domain that specifically binds to CD16A, and can specifically bind to 4-1BB, CLDN18.2, and CD16A simultaneously or separately.

[0096] In some embodiments, when the second antigen-binding domain that specifically binds to CLDN18.2 does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB does not activate 4-1BB signaling, or binds to the 4-1BB antigen but does not activate 4-1BB signaling.

[0097] In the above embodiments, the first antigen-binding domain that specifically binds to 4-1BB, the second antigen-binding domain that specifically binds to CLDN18.2, and the third antigen-binding domain that specifically binds to CD16A can be independently selected from, for example, Fab, Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (eg, VH or VL or VHH), and scFv. For example, the first antigen-binding domain that specifically binds to 4-1BB, the third antigen-binding domain that specifically binds to CD16A is VHH, and the second antigen-binding domain that specifically binds to CLDN18.2 is Fab.

[0098] Regarding the first antigen-binding domain that specifically binds to 4-1BB:

[0099] In some embodiments, the first antigen-binding domain that specifically binds to 4-1BB in the CLDN18.2 / 4-1BB binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of any one of the amino acid sequences shown in SEQ ID NOs: 10, 18-21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. The immunoglobulin single variable structure specifically binds to the 4-1BB antigen or a fragment thereof.

[0100] In some embodiments, the immunoglobulin single variable domain comprises any one or any combination of the above-mentioned CDR1, CDR2 and CDR3.

[0101] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 11, 12, and 13, respectively, or in SEQ ID NOs: 11, 12, and 22, respectively. These are CDRs defined according to the Kabat numbering system.

[0102] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are as shown in GFTFSSYA (SEQ ID NO: 66), INSGGEST (SEQ ID NO: 67), AKHPLTFTIATMNDYDY (SEQ ID NO: 68), or as shown in SEQ ID NOs: 66, 67, and AKHPLTYTIATMNDYDY (SEQ ID NO: 69), respectively. These are CDRs defined according to the IMGT numbering system.

[0103] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown as GFTFSSY (SEQ ID NO: 70), NSGGES (SEQ ID NO: 71), SEQ ID NO: 13, or as shown in SEQ ID NOs: 70, 71, and 22, respectively. These are CDRs defined according to the Chothia numbering system.

[0104] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown as GFTFSSYAMS (SEQ ID NO: 72), DINSGGESTF (SEQ ID NO: 73), SEQ ID NO: 13, or as shown in SEQ ID NOs: 72, 73, and 22, respectively. These are CDRs defined according to the AbM numbering system.

[0105] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are as shown in SSYAMS (SEQ ID NO: 74), WVSDINSGGESTF (SEQ ID NO: 75), AKHPLTFTIATMNDYD (SEQ ID NO: 76), or as shown in SEQ ID NOs: 74, 75, and AKHPLTYTIATMNDYD (SEQ ID NO: 77), respectively. These are CDRs defined according to the Contact numbering system.

[0106] In some embodiments, the immunoglobulin single variable domain is humanized, affinity matured, engineered to remove / reduce T cell epitopes, reduce antibody deamidation, and / or reduce antibody isomerization.

[0107] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing T cell epitopes, and has one or more changes in one or more CDRs, which result in reduced immunogenicity of the binding protein.

[0108] In some embodiments, the immunoglobulin single variable domain is humanized. The heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-64*04, IGHV3-23*03, and / or IGHV3-74*01. In some embodiments, FR1 is derived from IGHV3-64*04, FR2 is derived from IGHV3-23*03, and FR3 is derived from IGHV3-74*01.

[0109] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 10, 18-21, or has at least 80% sequence identity with any one of SEQ ID NOs: 10, 18-21.

[0110] In some embodiments, the first antigen binding domain that specifically binds to 4-1BB in the CLDN18.2 / 4-1BB binding protein comprises the aforementioned 4-1BB binding protein of the present disclosure, or comprises Urelumab, Utomilumab, ADG106 and WO2005035584A, WO2019037711A, US20190055314A, WO2019014328A3, US20210206867A, or an anti-4-1BB antibody or antigen-binding fragment thereof. The present disclosure incorporates the above-mentioned patents in their entirety.

[0111] Regarding the second antigen-binding domain that specifically binds to CLDN18.2:

[0112] In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 in the CLDN18.2 / 4-1BB binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0113] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 64. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems, for example, according to the Kabat numbering system.

[0114] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 57, 58, and 59, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 60, 61, and 62. These CDRs are defined according to the Kabat numbering system.

[0115] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in GYTFTSYW (SEQ ID NO: 78), IHPNSGST (SEQ ID NO: 79), and ARLKTGNSFDY (SEQ ID NO: 80), and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in QSLLNSGNQKNY (SEQ ID NO: 81), WA, and SEQ ID NO: 62. These CDRs are defined according to the IMGT numbering system.

[0116] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in GYTFTSY (SEQ ID NO: 82), HPNSGS (SEQ ID NO: 83), and SEQ ID NO: 59, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 60, 61, and 62. These CDRs are defined according to the Chothia numbering system.

[0117] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in GYTFTSYWMH (SEQ ID NO: 84), MIHPNSGSTN (SEQ ID NO: 85), and SEQ ID NO: 59, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 60, 61, and 62. These CDRs are defined according to the AbM numbering system.

[0118] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in TSYWMH (SEQ ID NO: 86), WMGMIHPNSGSTN (SEQ ID NO: 87), and ARLKTGNSFD (SEQ ID NO: 88), and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in LNSGNQKNYLTWY (SEQ ID NO: 89), LLIYWASTRE (SEQ ID NO: 90), and QNAYTYPF (SEQ ID NO: 91). These CDRs are defined according to the Contact numbering system.

[0119] In some embodiments, the VH that specifically binds to the second antigen-binding domain of CLDN18.2 comprises an amino acid sequence as shown in SEQ ID NO: 63, or at least 80% or at least 90% identical thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 64, or at least 80% or at least 90% identical thereto.

[0120] In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, or IgG4.

[0121] In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 45, or is at least 80% or at least 90% identical thereto, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 46, or is at least 80% or at least 90% identical thereto.

[0122] This disclosure incorporates in its entirety the anti-CLDN18.2 antibodies of WO2020200196A, and such antibodies or antigen-binding fragments thereof may be used as the second antigen-binding domain specifically binding to CLDN18.2 of this disclosure. In some other embodiments, the second antigen-binding domain specifically binding to CLDN18.2 comprises the anti-CLDN18.2 antibodies or antigen-binding fragments of WO2021027850A, WO2014146672A, WO2021025177A, WO2016180782A, or WO2021254481A, which are also incorporated in their entirety by this disclosure.

[0123] Regarding the third antigen-binding domain that specifically binds to CD16A:

[0124] In some embodiments, the third antigen-binding domain that specifically binds to CD16A in the CLDN18.2 / 4-1BB binding protein comprises an immunoglobulin single variable domain, which comprises CDR1, CDR2, and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 23, 35-39, or CDR1, CDR2, and CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 24, 40-43; wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

[0125] In some embodiments, the immunoglobulin single variable domain comprises any one or any combination of the above-mentioned CDR1, CDR2 and CDR3.

[0126] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 25, 26, and 27, respectively, which are CDRs defined according to the Kabat numbering system.

[0127] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown as GFAFSTYA (SEQ ID NO: 92), INSDGSST (SEQ ID NO: 93), and AKGWISSPVWGDYVPPV (SEQ ID NO: 94), respectively, which are CDRs defined according to the IMGT numbering system.

[0128] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown as GFAFSTY (SEQ ID NO: 95), NSDGSS (SEQ ID NO: 96), and SEQ ID NO: 27, respectively, which are CDRs defined according to the Chothia numbering system.

[0129] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown as GFTFSTYAMY (SEQ ID NO: 97), TINSDGSSTR (SEQ ID NO: 98), and SEQ ID NO: 27, respectively, which are CDRs defined according to the AbM numbering system.

[0130] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown as STYAMY (SEQ ID NO: 99), WVSTINSDGSSTR (SEQ ID NO: 100), and AKGWISSPVWGDYVPP (SEQ ID NO: 101), respectively, which are CDRs defined according to the Contact numbering system.

[0131] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 28, 29, and 30, respectively. These are CDRs defined according to the Kabat numbering system. These are CDRs defined according to the Kabat numbering system.

[0132] In some embodiments, the immunoglobulin single variable domain is humanized, affinity matured, engineered to remove / reduce T cell epitopes, reduce antibody deamidation, and / or reduce antibody isomerization.

[0133] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing T cell epitopes, and has one or more changes in one or more CDRs, which result in reduced immunogenicity of the binding protein.

[0134] In some embodiments, the immunoglobulin single variable domain is humanized. The heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-23*04 or IGHV3-20*04. In some embodiments, when the parent immunoglobulin single variable domain is SEQ ID NO: 23, the heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-23*04; when the parent immunoglobulin single variable domain is SEQ ID NO: 24, the heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-20*04.

[0135] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 23, 35-39, or has at least 80% or at least 90% sequence identity thereto; or, as shown in any one of SEQ ID NOs: 24, 40-43, or has at least 80% or at least 90% sequence identity thereto.

[0136] In some embodiments, the third antigen-binding domain that specifically binds to CD16A comprises the aforementioned CD16A binding protein of the present disclosure, or the anti-CD16A antibody or antigen-binding fragment thereof in WO2006125668A, WO2007009065A, or WO2016177846A. The present disclosure incorporates the aforementioned patents in their entirety.

[0137] The following exemplary embodiments provide the CLDN18.2 / 4-1BB binding proteins of the present disclosure:

[0138] In some embodiments, the first antigen-binding domain that specifically binds to 4-1BB in the CLDN18.2 / 4-1BB binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence of any one of SEQ ID NOs: 10, 18-21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are as shown in SEQ ID NOs: 11, 12, and 13, respectively, or as shown in SEQ ID NOs: 11, 12, and 22, respectively. In some specific embodiments, the amino acid sequence of the immunoglobulin single variable domain is as shown in SEQ ID NOs: 10, 18-21, respectively, or has at least 80% or at least 90% sequence identity with any one of SEQ ID NOs: 10, 18-21. In some embodiments, the immunoglobulin single variable domain is a single domain antibody or VHH.

[0139] In some embodiments, the second antigen-binding domain of the CLDN18.2 / 4-1BB binding protein that specifically binds to CLDN18.2 comprises a VH and a VL. In some specific embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises the LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 64. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems, for example, according to the Kabat numbering system. In some specific embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 57, 58, and 59, and the VL comprises the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 60, 61, and 62. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 63, or at least 80% or at least 90% identical thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 64, or at least 80% or at least 90% identical thereto. In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the second antigen-binding domain that specifically binds to CLDN18.2 comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 45, or at least 80% or at least 90% identical thereto, and the light chain comprising an amino acid sequence as set forth in SEQ ID NO: 46, or at least 80% or at least 90% identical thereto.

[0140] Optionally, in some embodiments, the CLDN18.2 / 4-1BB binding protein further comprises a third antigen-binding domain that specifically binds to CD16A. In some specific embodiments, the third antigen-binding domain that specifically binds to CD16A comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of any one of the amino acid sequences set forth in SEQ ID NOs: 23, 35-39, or CDR1, CDR2, and CDR3 of any one of the amino acid sequences set forth in SEQ ID NOs: 24, 40-43; wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are as shown in SEQ ID NOs: 25, 26, and 27, or as shown in SEQ ID NOs: 28, 29, and 30, respectively. In some embodiments, the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 23, 35-39, or has at least 80% or at least 90% sequence identity thereto, or as shown in any one of SEQ ID NOs: 24, 40-43, or has at least 80% or at least 90% sequence identity thereto. In some embodiments, the immunoglobulin single variable domain is a single domain antibody or VHH.

[0141] In some embodiments, the CLDN18.2 / 4-1BB binding protein has one or more (e.g., 2, 3, 4, 5, 6) first antigen binding domains that specifically bind to 4-1BB, and / or, one or more (e.g., 2, 3, 4) second antigen binding domains that specifically bind to CLDN18.2; optionally, there are further one or more (e.g., 2, 3, 4, 5, 6) third antigen binding domains that specifically bind to CD16A. In some specific embodiments, the CLDN18.2 / 4-1BB binding protein has two first antigen binding domains that specifically bind to 4-1BB, and two second antigen binding domains that specifically bind to CLDN18.2. In some specific embodiments, the CLDN18.2 / 4-1BB binding protein has two first antigen binding domains that specifically bind to 4-1BB, and two second antigen binding domains that specifically bind to CLDN18.2, and two third antigen binding domains that specifically bind to CD16A.

[0142] In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the valency ratio of the first antigen-binding domain that specifically binds to 4-1BB to the second antigen-binding domain that specifically binds to CLDN18.2 is between 6:1 and 1:3 (e.g., 4:1 to 1:2), for example, 1:1, 1:2, 2:1, 1:3, or 3:1. In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the valency ratio of the first antigen-binding domain that specifically binds to 4-1BB to the second antigen-binding domain that specifically binds to CLDN18.2 to the third antigen-binding domain that specifically binds to CD16A is between 6:3:1 and 1:3:6, for example, 1:1:1, 1:2:2, 2:2:1, 2:1:2, 1:2:1, 3:1:3, 3:1:2, 2:1:3, 3:1:1, or 3:2:1.

[0143] In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the first antigen-binding domain that specifically binds to 4-1BB is located at the N-terminus and / or C-terminus of the second antigen-binding domain that specifically binds to CLDN18.2; alternatively, in some embodiments, the third antigen-binding domain that specifically binds to CD16A is located at the N-terminus and / or C-terminus of the second antigen-binding domain that specifically binds to CLDN18.2.

[0144] In some embodiments, the CLDN18.2 / 4-1BB binding protein further comprises a human immunoglobulin Fc region, for example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region.

[0145] In some embodiments, the Fc region can enable the binding protein to form a dimeric molecule.

[0146] In some embodiments, the Fc region comprises a mutation that extends the in vivo half-life, which depends on the FcRn binding affinity. The extension of half-life can allow a reduction in the amount of drug administered to the patient and / or a reduction in the frequency of administration. For example, the Fc region comprises an M252Y, S254T and / or T256E mutation.

[0147] In some embodiments, the Fc region is an Fc region with enhanced effector function, e.g., an Fc region with enhanced ADCC, ADCP, and / or CDC; or an Fc region with reduced fucosylation. The Fc region can have mutations. Exemplary IgG1 Fc regions with enhanced effector function include those with the following substitutions or any combination thereof: S239D, S239E, S239K, F241A, V262A, V264D, V264L, V264A, V264S, D265A, D265S, D265V, F296A, Y296A, R301A, I332E, S239D / I332E, S239D / A330S / I332E, S239D / A330L / I332E, S298A / D333A / K334A, P247I / A339D, P247I / A339Q, D280H / K290S, D 396L, K290E / S298G / T299A, K290N / S298G / T299A, K290E / S298G / T299A / K326E, or K290N / S298G / T299A / K326E, or any combination of the foregoing positions. In some specific embodiments, the CLDN18.2 / 4-1BB binding protein contains a human IgG1 Fc region with S239D / I332E mutations.

[0148] In other specific embodiments, the Fc region can be an Fc region with reduced effector function, for example, the Fc region can have a mutation, and exemplary IgG Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0149] In the present disclosure, Fc regions with enhanced effector function are selected in CLDN18.2 / 4-1BB binding proteins (e.g., anti-CLDN18.2 / 4-1BB bispecific antibodies, anti-CLDN18.2 / 4-1BB / CD16A trispecific antibodies), which are significantly superior to Fc regions with unchanged or reduced effector function.

[0150] In some specific embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 65. In other specific embodiments, the Fc comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-16.

[0151] In some embodiments, the Fc region of the CLDN18.2 / 4-1BB binding protein comprises a first subunit Fc1 and a second subunit Fc2 that are capable of associating with each other.

[0152] In some embodiments, Fc1 and Fc2 contain amino acid mutations that result in Fc1 preferentially pairing with Fc2 or forming heterodimers with Fc2 compared to Fc1. In some embodiments, the mutations are located in the CH3 of Fc1 and Fc2. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater electrostatic complementarity than a wild-type mutant lacking the mutations. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater steric complementarity than a wild-type mutant lacking the mutations.

[0153] In some embodiments, in Fc1 and Fc2, for example, within the CH3 / CH3 interface, one or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby generating a protrusion (or knob) on the surface of the CH3 domain of Fc1, and one or more, preferably two or three, amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby generating a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some embodiments, the import residue with a larger side chain volume is phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W). In some embodiments, the import residue with a smaller side chain volume is serine (S), alanine (A), valine (V), or threonine (T).

[0154] In some specific embodiments, the Fc1 comprises at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (hole mutation modification), and the Fc2 comprises T366W (knob mutation modification); or the Fc1 comprises T366W (knob mutation modification), and the Fc2 comprises at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (hole mutation modification).

[0155] In some embodiments, Fc1 and Fc2, for example, CH3, may contain a natural non-cysteine ​​to cysteine ​​mutation, such as S354C in Fc1 and Y349C in Fc2; or Y349C in Fc1 and S354C in Fc2.

[0156] In some embodiments, Fc1 and Fc2, for example, in the Fc1CH3 / Fc2CH3 interface, comprise the following amino acid mutations or combinations thereof: T366Y / Y407T; T366W / Y407A; T366Y / Y407T; T394W / F405A; T366Y / F405AT394W / Y407T; T366W / F405WT394S / Y407A; F405W / T394S; D399C / K392C; T366W / T366S / L368A / Y407V; T366W / D399C / T366S / L368A / K392C / Y407V; T366W / K392C / T366S / D399C / L368A / Y407V; S354C / T366W / Y349C / T366S / L36 8A / Y407V; Y349C / T366W / S354C / T366S / L368A / Y407V; E356C / T366W / Y349C / T366S / L368A / Y407V; Y349C / T366W / E356C / T366S / L368A / Y407V; E357C / T366W / Y349C / T366S / L368A / Y407V; and Y349C / T366W / E357C / T366S / L368A / Y407V.

[0157] In some embodiments, Fc1 and Fc2 further comprise amino acid mutations that form an electrostatic interaction interface between Fc1 and Fc2 (e.g., CH3 and CH3). Amino acid mutations that form an electrostatic interaction interface are, for example, selected from the following: K370E / D399K / K439D / D356K / E357K / K409D; K409D / D399K; K409E / D399K; K409E / D399R; K409D / D399R; D339K / E356K; D399K / E356K / K409D / K392D; D 399K / E356K / K409D / K439D; D399K / E357K / K409D / K370D; D399K / E356K / E357K / K409D / K 392D / K370D; D399K / E357K / K409D / K392D; K392D / K409D / D399K; and K409D / K360D / D399K.

[0158] In some embodiments, Fc1 and / or Fc2 comprise domains from different antibody subtypes, such as CH3 from different antibody subtypes.

[0159] In addition, the present disclosure cites WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012058768, WO2013157954, and WO2013096291 for modifying the CH3 region of the Fc region to enhance heterodimerization.

[0160] In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the first antigen-binding domain that specifically binds to 4-1BB and the second antigen-binding domain that specifically binds to CLDN18.2 are directly or via a linker. In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the first antigen-binding domain that specifically binds to 4-1BB, the second antigen-binding domain that specifically binds to CLDN18.2, and the third antigen-binding domain that specifically binds to CD16A are directly or via a linker. In some embodiments, the first antigen-binding domain that specifically binds to 4-1BB is directly or via a linker connected to the Fc region. In some embodiments, the third antigen-binding domain that specifically binds to CD16A is directly or via a linker connected to the light chain constant region of the second antigen-binding domain that specifically binds to CLDN18.2.

[0161] In some specific embodiments, the linker includes but is not limited to (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h The amino acid sequence shown, wherein m, n are each independently selected from integers of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), and h is independently selected from integers of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). In some embodiments, the linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structure. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, for example (G4S)2, (G4S)3.

[0162] In some specific embodiments, the CLDN18.2 / 4-1BB binding protein comprises a first polypeptide chain and a second polypeptide chain, and the first and second polypeptide chains are as follows from N-terminus to C-terminus:

[0163] (1) First polypeptide chain: [VH that specifically binds to the second antigen-binding domain of CLDN18.2]-CH1-Fc region-[Linker 1]a-[First antigen-binding domain that specifically binds to 4-1BB];

[0164] Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of CLDN18.2] -CL

[0165] (2) First polypeptide chain: [first antigen-binding domain that specifically binds to 4-1BB]-[linker 1]a-[VH of the second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region;

[0166] Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of CLDN18.2] -CL

[0167] (3) First polypeptide chain: [VH]-CH1-Fc region that specifically binds to the second antigen-binding domain of CLDN18.2;

[0168] Second polypeptide chain: [first antigen-binding domain that specifically binds to 4-1BB]-[Linker 1]a-[VL that specifically binds to the second antigen-binding domain of CLDN18.2]-CL

[0169] (4) First polypeptide chain: [VH]-CH1-Fc region that specifically binds to the second antigen-binding domain of CLDN18.2;

[0170] Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of CLDN18.2]-[Linker 1]a-CL [first antigen-binding domain that specifically binds to 4-1BB]

[0171] (5) First polypeptide chain: [VH that specifically binds to the second antigen-binding domain of CLDN18.2]-CH1-Fc region-[Linker 2]b-[First antigen-binding domain that specifically binds to 4-1BB];

[0172] Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of CLDN18.2]-[Linker 1]a-CL [first antigen-binding domain that specifically binds to 4-1BB]

[0173] (6) First polypeptide chain: [VH that specifically binds to the second antigen-binding domain of CLDN18.2]-CH1-Fc region-[Linker 3]c-[first antigen-binding domain that specifically binds to 4-1BB];

[0174] Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of CLDN18.2]-CL-[Linker 4]d-[Third antigen-binding domain that specifically binds to CD16A]

[0175] (7) First polypeptide chain: [VH that specifically binds to the second antigen-binding domain of CLDN18.2]-CH1-Fc region-[Linker 3]c-[Third antigen-binding domain that specifically binds to CD16A];

[0176] Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of CLDN18.2]-CL-[Linker 4]d-[First antigen-binding domain that specifically binds to 4-1BB]

[0177] Wherein, - represents a peptide bond, and a linker is a polypeptide capable of achieving a linking function. Linker 1, Linker 2, Linker 3, and Linker 4 may be the same or different; a, b, c, and d may be independently selected from 1 or 0, for example, a, b, c, and d are all 1. The linker is, for example, independently selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, and ASGS, for example, (G4S)2 and (G4S)3.

[0178] In some embodiments, a CLDN18.2 / 4-1BB binding protein is provided, comprising a first and a second polypeptide chain. In some specific embodiments, the amino acid sequence of the first polypeptide chain is as shown in any one of SEQ ID NOs: 47-49, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 46, or has at least 80% or at least 90% sequence identity thereto.

[0179] In some specific embodiments, the amino acid sequence of the first polypeptide chain is as shown in any one of SEQ ID NOs: 50, 52, and 53, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NOs: 51 or 54, or has at least 80% or at least 90% sequence identity thereto; for example, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 50 or 52, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 51, or has at least 80% or at least 90% sequence identity thereto; for example, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 53, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 54, or has at least 80% or at least 90% sequence identity thereto.

[0180] In some specific embodiments, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 55 or 56, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 46, or has at least 80% or at least 90% sequence identity thereto.

[0181] In some embodiments, a CLDN18.2 / 4-1BB binding protein is provided, comprising a first and a second polypeptide chain selected from the group consisting of:

[0182] (1) the amino acid sequence of the first polypeptide chain is as shown in any one of SEQ ID NOs: 47-49, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 46;

[0183] (2) the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 50 or 52, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 51;

[0184] (3) the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 53, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 54;

[0185] (4) The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 55 or 56, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 46.

[0186] In some embodiments, the CLDN18.2 / 4-1BB binding protein of the present disclosure has at least one activity selected from the following:

[0187] (a)≤10 -7 K D The value binds to human 4-1BB or its epitope;

[0188] (b) when not cross-linked with CLDN18.2 (or not bound to CLDN18.2), the 4-1BB signaling pathway is weakly activated or not activated, for example, at a concentration of 100 nM of antibody, the degree of activation when not cross-linked with CLDN18.2 (or not bound to CLDN18.2) does not exceed 10% of the activity under saturating antibody concentration conditions when cross-linked with CLDN18.2 (or bound to CLDN18.2);

[0189] (c) When cross-linked with CLDN18.2 (or not bound to CLDN18.2), the 4-1BB signaling pathway is strongly activated or strongly activated, for example, EC 50 Less than 1nM;

[0190] (d) activating T cells and / or promoting T cell proliferation;

[0191] (e) inhibiting tumor growth;

[0192] (f) having enhanced ADCC and / or ADCP functions.

[0193] For example, the detection of activation of the 4-1BB signaling pathway in (b) and (c) can be found in the 4-1BB / NF-κB luciferase reporter gene detection method of Example 2.

[0194] In some embodiments, the CLDN18.2 / 4-1BB binding proteins of the present disclosure are capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0195] In some embodiments, the CLDN18.2 / 4-1BB binding protein disclosed herein encompasses variants, wherein the variant has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to the first and second polypeptide chains in any combination of (1) to (4) above; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function.

[0196] In some embodiments, a CLDN18.2 / 4-1BB binding protein is provided that binds to or competes for binding to CLDN18.2 and / or 4-1BB, or binds to or competes for binding to the same epitope of CLDN18.2 and / or 4-1BB as the aforementioned CLDN18.2 / 4-1BB binding protein of the present disclosure. When the CLDN18.2 / 4-1BB binding protein further binds to CD16A, a CLDN18.2 / 4-1BB binding protein is provided that binds to or competes for binding to CLDN18.2 and / or 4-1BB and / or CD16A, or binds to or competes for binding to the same epitope of CLDN18.2 and / or 4-1BB and / or CD16A as the aforementioned CLDN18.2 / 4-1BB binding protein of the present disclosure.

[0197] In some embodiments, a CLDN18.2 / 4-1BB binding protein is provided that blocks the binding of the aforementioned CLDN18.2 / 4-1BB binding protein of the present disclosure to CLDN18.2 and / or 4-1BB; optionally, blocks the binding of the aforementioned CLDN18.2 / 4-1BB binding protein of the present disclosure to CD16A.

[0198] In some embodiments, a protein or molecule is provided, comprising any of the aforementioned CLDN18.2 / 4-1BB binding proteins disclosed herein. For example, the protein or molecule is a conjugate, which may comprise any detectable label.

[0199] Polynucleotides and vectors

[0200] The present disclosure provides polynucleotides encoding the 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein of the present disclosure. The nucleic acid of the present disclosure may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the nucleic acid of the present disclosure is a substantially isolated nucleic acid.

[0201] The nucleic acid of the present disclosure may also be in the form of a vector, may be present in a vector and / or may be a part of a vector, such as a plasmid, a cosmid, a YAC or a viral vector. The vector may be an expression vector in particular, i.e., a vector for expressing 4-1BB-associated proteins, CD16A-associated proteins, CLDN18.2 / 4-1BB-associated proteins in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector generally comprises at least one nucleic acid of the present disclosure, which is operably connected to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common sense for those skilled in the art to select the elements and their sequences for expression in a specific host. 4-1BB-associated proteins, CD16A-associated proteins, CLDN18.2 / 4-1BB-associated proteins of the present disclosure are useful or necessary regulatory elements and other elements, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0202] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0203] host cells

[0204] The present disclosure provides recombinant host cells that express or are capable of expressing one or more 4-1BB binding proteins, CD16A binding proteins, CLDN18.2 / 4-1BB binding proteins of the present disclosure and / or contain polynucleotides or vectors of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0205] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0206] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0207] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0208] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0209] Preparation method

[0210] The present disclosure provides a method for preparing 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, comprising: expressing the target protein in a host cell as described above, and isolating the target protein from the host cell. Optionally, a purification step may also be included, for example, purifying with an A or G Sepharose FF column containing an adjusted buffer, washing away non-specifically bound components, eluting the bound antibodies with a pH gradient method, detecting with SDS-PAGE, and collecting. Alternatively, filtration and concentration are performed using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product must be immediately frozen, such as at -70°C, or lyophilized.

[0211] Methods for producing and purifying antibodies are well known in the art and can be found in, for example, the Cold Spring Harbor Manual of Antibody Laboratory Techniques (Chapters 5-8 and 15).

[0212] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0213] Composition

[0214] The present disclosure provides a composition comprising the aforementioned 4-1BB binding protein, CD16A binding protein, and CLDN18.2 / 4-1BB binding protein of the present disclosure. For example, a pharmaceutical composition is provided, which contains an effective amount of the aforementioned 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein for treating, alleviating or preventing cancer, and at least one pharmaceutically acceptable excipient, diluent or carrier.

[0215] In some specific embodiments, the unit dosage of the pharmaceutical composition may contain 0.01 to 99 weight % of 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, or the amount of 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein in a unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0216] In some embodiments, there is provided a product or product (such as a test kit), which comprises at least one container, wherein the container independently comprises the aforementioned 4-1BB-binding protein, CD16A-binding protein or CLDN18.2 / 4-1BB-binding protein. Alternatively, the product comprises a container and a label. The container is such as a bottle, a syringe and a test tube. The container holds a composition effective for treating a disease. A label on or connected to the container indicates that the composition is used to treat a selected disease.

[0217] Methods of treatment and pharmaceutical uses

[0218] The present disclosure provides methods for using the aforementioned 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, polynucleotides, and compositions (including pharmaceutical compositions) for treating, alleviating, preventing, and diagnosing diseases or disorders.

[0219] In some embodiments, a method for improving, alleviating, treating or preventing a disease is provided, comprising administering to a subject an effective amount of the aforementioned 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, polynucleotide, composition (including pharmaceutical composition) for improvement, alleviation, treatment or prevention.

[0220] In some embodiments, the present invention provides a use of the 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein polynucleotide, and composition (including pharmaceutical composition) for preparing a drug for improving, alleviating, treating, or preventing a disease.

[0221] In some embodiments, the aforementioned disease is a proliferative disorder or any other disease or disorder characterized by uncontrolled cell growth, such as cancer.In this disclosure, cancer and tumor are used interchangeably.

[0222] In some embodiments, the aforementioned cancer is a solid tumor or a hematological tumor.

[0223] In some embodiments, the aforementioned cancer is advanced or metastatic.

[0224] In some embodiments, the aforementioned disease is CLDN18.2-related or CLDN18.2-positive, such as CLDN18.2-positive cancer. In some embodiments, diseases related to Claudin18.2 or CLDN18.2-positive can be diagnosed by detecting or measuring cells expressing Claudin18.2 using the antibodies or antibody fragments disclosed herein. In order to detect cells expressing polypeptides, known immunoassay methods can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc. can be used. In addition, fluorescent antibody staining using the FMAT8100HTS system (Applied Biosystem) can be used. In the present disclosure, there is no particular limitation on the sample to be tested for detecting or measuring the target antigen (e.g., Claudin18.2), as long as it has the possibility of containing cells expressing the target antigen (e.g., Claudin18.2), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

[0225] In some embodiments, the aforementioned cancer is selected from the following or a combination thereof: lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematological cancer, or any other disease or condition characterized by uncontrolled cell growth.

[0226] Detection

[0227] The present disclosure provides detection uses of 4-1BB binding proteins, CD16A binding proteins, CLDN18.2 / 4-1BB binding proteins, polynucleotides, and compositions. The present disclosure also provides methods, systems, or devices for in vivo or in vitro detection of 4-1BB, CLDN18.2, and CD16A, comprising treating a sample with the aforementioned binding proteins, polynucleotides, and compositions disclosed herein.

[0228] In some embodiments, an in vitro detection method, system, or device may include, for example:

[0229] (1) contacting a sample with the 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, polynucleotide, or composition disclosed herein;

[0230] (2) detecting a complex formed between the aforementioned binding protein, polynucleotide, and sample; and / or

[0231] (3) contacting a reference sample (e.g., a control sample) with the binding protein and nucleic acid; and

[0232] (4) Determining the extent of complex formation by comparison with a reference sample. A change (e.g., a statistically significant change) in complex formation in the sample compared to the control sample indicates the presence of 4-1BB, CLDN18.2, and CD16A in the sample.

[0233] In some embodiments, a kit is also provided, comprising the aforementioned 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, polynucleotides, and diagnostic instructions. The kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the 4-1BB binding protein, CD16A binding protein, and CLDN18.2 / 4-1BB binding protein can be formulated as a pharmaceutical composition.

[0234] Definition of terms

[0235] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0236] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0237] "CLDN18 protein" or "CLDN18" is a protein encoded by the Claudin18 gene in humans and belongs to the family of tight junction proteins. Claudin-18 can control the flow of molecules between layer cells. The Claudin-18 protein structure includes four transmembrane regions, two extracellular loops, and its N-terminus and C-terminus are in the cytoplasm. Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, and the sequences of the two differ only by eight amino acids in the first extracellular loop. The expression distribution of Claudin 18.1 and Claudin18.2 is different. Claudin 18.1 is selectively expressed in normal lung cells, and Claudin 18.2 is highly restricted in normal cells, but is frequently ectopically activated and overexpressed in various tumors (gastric cancer, lung cancer, pancreatic cancer, etc.).

[0238] "CD16," also known as FcγRIII, is a low-affinity receptor for the Fc fragment of IgG involved in antibody-dependent cellular cytotoxicity (ADCC). Human FcγRIII has two subtypes: FcγRIIIA (i.e., CD16A) and FcγRIIIB (i.e., CD16B), whose extracellular immunoglobulin-binding domain sequences share 96% sequence identity (van de Winkel and Capel, 1993, Immunol Today 14(5): 215-221). CD16A is a transmembrane receptor expressed on macrophages, mast cells, and NK cells. On NK cells, the α chain of CD16A binds to the immunoreceptor tyrosine-based activation motif (ITAM) containing the FcεRIγ-chain and / or the T cell receptor (TCR) / CD3ζ-chain to induce signal transduction (Wirthmueller et al., 1992, J. Exp. Med. 175:1381-1390), leading to cytokine production and cytotoxicity. CD16B is present on polymorphonuclear granulocytes (PMNs) as a glycosylphosphatidylinositol (GPI)-anchored receptor (FcγRIIIB subtype), which is unable to induce tumor cell cytotoxicity (van de Winkel and Capel, 1993, supra). In addition, CD16B is present in serum as a soluble receptor, which, once bound to antibodies in vivo, may cause side effects by forming immune complexes.

[0239] "4-1BB protein" or "4-1BB" is also known as CD137, tumor necrosis factor receptor superfamily 9, a member of the TNF receptor superfamily (TNFRSF), which is expressed on CD8 + and CD4 +Costimulatory molecules on the surface of cells such as T cells, regulatory T cells (Tregs), NK cells and NKT cells, B cells and neutrophils. 4-1BB is a costimulatory molecule that is expressed after immune cells are activated. The NCBI accession number for human 4-1BB protein is NP_001552.2. In the present disclosure, "4-1BB" may optionally include any such protein or fragment or variant thereof, including but not limited to known or wild-type 4-1BB as described in the present disclosure, and any naturally occurring splice variant, amino acid variant or isoform, such as human 4-1BB shown in SEQ ID NO: 11.

[0240] "4-1BB binding protein" covers any protein capable of specifically binding to 4-1BB or any molecule thereof, including but not limited to antibodies, antigen-binding fragments thereof, or conjugates thereof as defined in the present disclosure for 4-1BB. The "4-1BB binding protein" of the present disclosure may include at least one (e.g., 1, 2, 3, 4, 5, 6 or more) immunoglobulin single variable domain (e.g., VHH) that binds to 4-1BB. The "4-1BB binding protein" of the present disclosure, in addition to the immunoglobulin single variable domain comprising 4-1BB, may also include a linker and / or a portion with effector function, such as a half-life extending portion (e.g., an immunoglobulin single variable domain that binds serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0241] “CD16A binding protein” adopts a definition similar to that of “4-1BB binding protein”, which covers any protein that can specifically bind to CD16A or any molecule thereof, including but not limited to antibodies, antigen-binding fragments thereof, or conjugates and fusion proteins thereof as defined in the present disclosure for CD16A.

[0242] "CLDN18.2 / 4-1BB binding protein" encompasses any molecule that can specifically bind to the CLDN18.2 protein or its epitope and the 4-1BB protein or its epitope, including but not limited to antibodies, polypeptides, fusion proteins of antibodies and polypeptides, or conjugates thereof. In some embodiments, the "CLDN18.2 / 4-1BB binding protein" encompasses the anti-CLDN18.2 / 4-1BB bispecific antibodies in the embodiments of the present disclosure. In some embodiments, the "CLDN18.2 / 4-1BB binding protein" of the present disclosure further comprises a CD16A binding domain, which can further bind to CD16A, for example, encompassing the anti-CLDN18.2 / 4-1BB / CD16A trispecific antibodies in the embodiments of the present disclosure.

[0243] "Antibodies" encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of antibody heavy and light chains vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0244] The antibodies disclosed herein may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in various embodiments. Generally speaking, the antibodies disclosed herein are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0245] "Antigen-binding fragments" encompass single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0246] For the determination or definition of CDRs, the definitive depiction of CDRs and the identification of residues in the binding site of the antibody can be completed by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).In addition, other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on the prediction or experimental results that a particular residue or residue group does not significantly affect antigen binding. As used in this disclosure, CDRs may refer to CDRs defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0247] Table 1. Relationships between CDR numbering systems

[0248] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0249] "Effector function" in the present disclosure refers to those biological activities that can be attributed to the Fc region of an antibody and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), Fc receptor (FcR) binding, cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, cell surface receptor (e.g., B cell receptor) downregulation and B cell activation. For example, ADCC is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind. "Enhanced ADCC" is defined as an enhancement of the number of target cells lysed within a given time at a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and / or a reduction in the antibody concentration in the medium surrounding the target cells required for the lysis of a given number of target cells within a given time by the ADCC mechanism. Enhancement of ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell but not engineered using the same standard production, purification, formulation, and storage methods known to those skilled in the art. For example, enhancement of ADCC mediated by an antibody comprising an amino acid substitution in its Fc region that enhances ADCC is relative to ADCC mediated by the same antibody without the amino acid substitution in the Fc region. Suitable assays for measuring ADCC are well known in the art (see, for example, WO2006 / 082515 or WO2012 / 130831).

[0250] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for specific functional properties of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0251] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.

[0252] An "immunoglobulin variable domain" is an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0253] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the complementarity determining regions (CDRs) of that variable domain.

[0254] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that is capable of forming a functional antigen binding site in the absence of interaction with other variable domains (e.g., in the absence of the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNARs, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are IgG1 or IgG2 domains. TM ) and (single domain) antibodies (such as dAbs) as the VL domain or derived from the VL domain TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0255] "VHH" is also called heavy chain single domain antibody, VHH, V HH domains, VHH antibody fragments, VHH antibodies, nanobodies, are variable domains of antigen-binding immunoglobulins known as "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). "VHH" is used to distinguish the variable domains from the heavy chain variable domains (referred to herein as "VH domains" or VH) and light chain variable domains (referred to herein as "VL domains" or VL) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for additional antigen-binding domains (this is in contrast to the VH or VL domains in conventional tetrapeptide chain structure antibodies, in which case the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. H H domain", "VHH antibody fragment", "VHH antibody", as well as" "Nanobody" is a trademark of Ablynx NV, Ghent, Belgium. VHHs include, but are not limited to, natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained by phage display technology. The total number of amino acid residues in a VHH will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94 / 04678.

[0256] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0257] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the human variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of non-human protein components. To avoid a simultaneous decrease in immunogenicity and activity, minimal reverse mutations can be performed on the fully human variable region to maintain activity. Examples of "humanization" include "humanizing" a Camelidae-derived VHH domain by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain human framework region sequences from IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), i.e., CDR "grafting" onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies that are further subjected to affinity maturation of CDRs by phage display. In addition, in order to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequences can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0258] An "affinity matured" antibody refers to an antibody that has one or more changes in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such changes, and such changes result in an improvement in the affinity of the antibody for the antigen. For example, an "affinity matured" 4-1BB binding protein or anti-4-1BB antibody has one or more changes in one or more CDRs that result in an increase in affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0259] Typically, the 4-1BB binding protein, CLDN18.2 / 4-1BB binding protein, CD16A binding protein of the present disclosure will be measured as in Biacore or KinExA or Fortibio assays, preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen or target protein to be bound (i.e. 4-1BB, CLDN18.2, CD16A). Any -4 M's K DValues ​​are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.

[0260] "Epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining the binding of an epitope to a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0261] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TMThe K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with those for non-target interactions (e.g., control antibodies known not to bind 4-1BB, CLDN18.2, or CD16A). D The value is evaluated.

[0262] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0263] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0264] "Nucleic acid" or "polynucleotide" are used interchangeably in this disclosure to refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and, in the case of single-stranded, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0265] "Host cell" includes individual cells or cell cultures that may be or have been recipients of vectors for incorporation of polynucleotide inserts. Host cells include the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0266] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0267] "Arresting the growth of" or "growth inhibition" refers to inhibiting the growth or proliferation of cells.

[0268] "Proliferative disease" refers to a condition associated with some degree of abnormal cell proliferation. In one embodiment, a proliferative condition refers to cancer. "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "proliferative disease," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0269] "Preventing cancer" refers to delaying, inhibiting, or preventing the onset of cancer in a subject in whom the onset of cancer or tumorigenesis has not been demonstrated, but in whom a predisposition to cancer has been identified, for example, by genetic screening or other methods. This also includes treating a subject with a precancerous condition to halt the progression of the precancerous condition to a malignant tumor or to cause its regression.

[0270] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0271] "Treatment" means administering an internal or external therapeutic agent, such as a binding protein or a pharmaceutical composition thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0272] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0273] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to."

[0274] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0275] FIG1 shows the FACS binding test results of the test antibody and human 4-1BB antigen.

[0276] FIG2 shows the 4-1BB / NF-κB luciferase reporter gene assay results of the test antibodies before and after FcγRIIb cross-linking.

[0277] Figure 3 shows the FACS detection results of the binding of anti-4-1BB antibody C5 and its humanized antibodies C5_V1, C5_V2, and C5_V3 to human 4-1BB on the surface of HEK293 cells, using Urelumab and isotype IgG1 antibodies as controls.

[0278] Figure 4 shows the results of the detection of the activation effect of the anti-4-1BB antibody C5 and its humanized antibodies C5_V1, C5_V2, and C5_V3 on the NF-κB signaling pathway, using Urelumab and isotype IgG1 antibodies as controls.

[0279] Figure 5 shows the FACS detection results of the binding of C5_V2 and TCE-removed C5_V2-YTI to human 4-1BB on the surface of HEK293 cells, using Urelumab and isotype IgG1 antibodies as controls.

[0280] Figure 6 shows the binding assay of anti-CD16A antibodies to cells overexpressing human CD16A, human CD16B, and cynomolgus monkey CD16. In the human CD16B-overexpressing cell binding assay, VHH-2-LALA, which binds to human CD16B, was used as a control (VHH-2 is from patent US20190276554). Isotype IgG was also used as a control in each experiment.

[0281] Figures 7A and 7B show the binding of anti-CD16A antibodies 34, 501 and humanized anti-CD16A antibodies 34H3, 501-V3NQ to cells overexpressing human CD16A or CD16B. In the cell binding experiment overexpressing human CD16B, VHH-2-LALA, which can bind to human CD16B, was used as a control (VHH-2 is from patent US20190276554). Isotype IgG was used as a control in each experiment.

[0282] Figure 8 is a schematic diagram of the structure of the CLDN18.2 / 4-1BB bispecific antibody.

[0283] Figure 9 is a schematic diagram of the structure of the CLDN18.2 / 4-1BB / CD16A trispecific antibody.

[0284] Figure 10 shows the binding test results of the C5_V2-YTI modified antibody 1903 x C5_V2-YTI-LALA to human 4-1BB on the surface of HEK293 cells, using 1903 x C5_V2-LALA, C5_V2, and isotype IgG1 as controls.

[0285] Figure 11 shows the activation test results of the NF-κB signaling pathway by the C5_V2-YTI modified antibody 1903 x C5_V2-YTI-LALA, using 1903 x C5_V2-LALA, TJ-CD4B, and isotype IgG1 as controls.

[0286] Figures 12A to 12C show the results of the binding activity assays of the CLDN18.2 / 4-1BB multifunctional antibodies 1903 x C5_V2 x 34H3 and 1903 x C5_V2 against cell surface CLDN18.2 / CD16A V176 / 4-1BB antigens. Figure 12A shows the results of the antibody binding assay to NUGC4-hi18.2 cells, using 1903, IMAB362, TJ-CD4B, and isotype IgG1 as controls; Figure 12B shows the results of the antibody binding assay to CHOK1-CD16A V176A cells, using IMAB362, TJ-CD4B, 34H-Fc, and isotype IgG1 as controls; and Figure 12C shows the results of the antibody binding assay to HEK293-Hu4-1BB cells, using TJ-CD4B, C5-_V2, and isotype IgG1 as controls.

[0287] Figures 13A to 13D show the results of NK cell-induced cytotoxicity against CLDN18.2-expressing target cells using the multifunctional antibodies 1903 x C5_V2-YTI x 34H3 and 1903 x C5_V2-YTI. IMAB362 and isotype IgG1 were used as controls. Figure 13A: ADCC of PBMC against NUGC4-hi18.2 cells (donor SC190061); Figure 13B: ADCC of PBMC against NUGC4-hi18.2 cells (donor S2001102); Figure 13C: ADCC of PBMC against SNU601 cells (donor SC190061); Figure 13D: ADCC of PBMC against SNU601 cells (donor S2001102).

[0288] Figure 14 shows the results of the macrophage phagocytosis test of tumor cells induced by the CLDN18.2 / 4-1BB multifunctional antibodies 1903 x C5_V2-YTI x 34H3 and 1903 x C5_V2-YTI, using IMAB362 and isotype IgG1 as controls.

[0289] Figure 15 shows the results of CLDN18.2-dependent 4-1BB / NF-κB luciferase reporter gene assays using the CLDN18.2 / 4-1BB multifunctional antibodies 1903 x C5_V2 x 34H3 and 1903 x C5_V2. The left side shows the results before CLDN18.2 cross-linking, and the right side shows the results after CLDN18.2 cross-linking. TJ-CD4B, ADG-106, and isotype IgG1 were used as controls.

[0290] Figures 16A and 16B show the activation test results of T lymphocytes from donors 1 and 2 by the CLDN18.2 / 4-1BB multifunctional antibodies 1903 x C5_V2-YTI x 34H3 and 1903 x C5_V2-YTI, respectively. TJ-CD4B and isotype IgG1 were used as controls.

[0291] Figure 17 shows the results of the NK cell killing test induced by the CLDN18.2 / 4-1BB multifunctional antibodies 1903 x C5_V2-YTI x 34H3 and 1903 x C5_V2-YTI, using isotype IgG1 as a control.

[0292] Figures 18A and 18B show the results of tumor inhibition testing of the CLDN18.2 / 4-1BB multifunctional antibodies 1903 x C5_V2-YTI x 34H3 and 1903 x C5_V2-YTI against MC38-hCLDN18.2 xenografts, using vehicle and TJ-CD4B as controls. Figure 18A shows tumor volume, and Figure 18B shows mouse body weight.

[0293] Figure 19 shows the effects of the CLDN18.2 / 4-1BB multifunctional antibodies 1903 x C5_V2-YTI x 34H3 and 1903 x C5_V2-YTI on mouse ALT / AST, using vehicle and TJ-CD4B as controls.

[0294] FIG20 shows the pharmacokinetic curve of the CLDN18.2 / 4-1BB multifunctional antibody 1903 x C5_V2 in h4-1BB transgenic mice, with 1903 used as a control. DETAILED DESCRIPTION

[0295] The following examples further illustrate the present disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0296] Proteins used in this disclosure: Human 4-1BB protein (Human 4-1BB / TNFSF9 Protein, His Tag, purchased from Acrobiosystems, Cat. No. 41B-H5227), Human 4-1BB protein (biotin / His tag) (Biotinylated Human 4-1BB / TNFRSF9 Protein, Avitag TM , His Tag, purchased from Acrobiosystems, product number 41B-H82E3), monkey 4-1BB protein (Cynomolgus / Rhesus macaque 4-1BB / TNFRSF9 Protein, His Tag, purchased from Acrobiosystems, product number 41B-C52H4), the starting and ending amino acid sequences are Leu24-Gln186.

[0297] The above protein reagents can be used in the experiments of various embodiments of the present disclosure, including as immunization antigens, screening antigens, and activity and function identification. The amino acid sequences of the control molecules used in the present disclosure are shown in Table 2.

[0298] Table 2. Control molecule sequences

[0299] Example 1. Screening and preparation of anti-4-1BB single domain antibodies

[0300] 1. Alpaca Immunization, Titer Testing, and Phage Library Affinity Panning

[0301] Alpacas were immunized with His-tagged human 4-1BB recombinant protein (Acrobiosystems, 41B-H5227) once every two weeks for a total of four immunizations. For the first immunization, 0.5 mg of antigen was mixed with 1 mL of Freund's complete adjuvant (CFA) and injected subcutaneously. For the next three immunizations, 0.25 mg of antigen was mixed with 1 mL of Freund's incomplete adjuvant (IFA) and injected subcutaneously. Blank serum was collected before immunization, and 50 mL of peripheral blood was collected one week after the third immunization and one week after the fourth immunization, PBMCs were separated, total RNA was extracted, purity was tested, reverse transcribed into DNA, and after two rounds of nested PCR, the nanoantibody target fragment was connected to the phage display vector. Phage library was obtained by electrotransfection.

[0302] >Human 4-1BB protein sequence

[0303] In order to obtain anti-4-1BB nanoantibodies that recognize both humans and monkeys, a two-round cross-screening strategy of human and monkey antigens was adopted. The first and second rounds of screening antigens used human 4-1BB and monkey 4-1BB, or monkey 4-1BB and human 4-1BB, respectively. Each round of screening used the Gly-HCl acid elution method to elute phages that specifically bind to 4-1BB. 96 clones (a total of 192 clones) were randomly selected from the first and second rounds of titer determination plates, and positive clones were screened by phage ELISA, and the optical density at 450nm was detected. The positive clones were sequenced. According to the sequencing results, sequence alignment and phylogenetic tree analysis were performed, and 14 unique sequences were screened out, including H27, H170, C3, C5, C145, etc., of which the C5 sequence is shown below.

[0304] >C5

[0305] (Note: The underlined area is the CDR region)

[0306] Table 3. CDRs of anti-4-1BB single domain antibody C5 (Kabat numbering convention)

[0307] 2. Expression and Purification of VHH-Fc Fusion Protein

[0308] The C5 sequence was linked to a human IgG1-Fc (SEQ ID NO: 16, underlined mutations) with C220A, S267E, and L328F mutations (numbered according to the Eu system). The sequence of the resulting VHH-Fc fusion protein is shown below. Furthermore, mutations such as L234A, L235A, and N297A (numbered according to the Eu system) were introduced into the Fc of human IgG1 to completely eliminate the antibody's FcγR-mediated effector function (e.g., as shown in SEQ ID NO: 15); and S228P (numbered according to the Eu system) was introduced into the Fc of human IgG4 to stabilize the antibody molecule and prevent half-molecule formation (e.g., as shown in SEQ ID NO: 16). In SEQ ID NOs: 14-16, underlined Fc mutations are shown.

[0309] >Human IgG1-Fc (containing C220A, S267E, L328F mutations)

[0310] >Human IgG1-Fc (containing C220A, L234A, L235A, N297A mutations)

[0311] >Human IgG4-Fc (containing S228P mutation)

[0312] The antibody sequence of C5 linked to SEQ ID NO: 14 is exemplified as shown in SEQ ID NO: 17.

[0313] (Note: Fc in italics)

[0314] Construct plasmids, transiently transfect cells, express antibodies, and purify them. After testing, the target antibody is obtained.

[0315] Example 2. Detection of Antigen Binding Activity and Agonist Activity of Anti-4-1BB Antibodies

[0316] 1. Detection of binding ability to 4-1BB antigen

[0317] The binding activity of anti-4-1BB single domain antibody to human 4-1BB protein was detected by flow cytometry.

[0318] HEK293-Hu4-1BB cells were obtained by transiently transfecting HEK293 cells (ATCC CRL-1573) with expression of human 4-1BB protein (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; purchased from Sino Biological, Cat#HG10041-ACR). The cell culture medium was DMEM (Gibco, Cat#11995065) containing 10% fetal bovine serum. The experimental culture medium was sterile PBS (phosphate buffered saline, pH 7.40) containing 2% fetal bovine serum. The HEK293-Hu4-1BB cells were washed twice with the experimental culture medium and 1×10 cells were plated per well. 5 HEK293-Hu4-1BB cells were seeded in 96-well U-bottom plates and tested with various concentrations of the anti-4-1BB antibody VHH-Fc sample. The cells were incubated at 4°C for 1 hour and washed twice with experimental medium. Goat anti-human IgG (H+L) Alexa Fluor 488 antibody (Thermo, Cat#A11013) was then added and washed twice before fluorescence signal readings were obtained by flow cytometry. Urelumab monoclonal antibody was used as a positive control. The MFI values ​​for each antibody are shown in Figure 1.

[0319] The results showed that the anti-4-1BB antibodies involved had different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, among which C5 had good cell membrane surface antigen binding activity.

[0320] 2. Detection of agonist activity on the 4-1BB signaling pathway

[0321] The agonist activity of anti-4-1BB antibodies was assessed using a 4-1BB / NF-κB reporter gene.

[0322] HEK293 cells (ATCC CRL-1573) were transiently transfected with the human 4-1BB gene (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; purchased from Sino Biological, Cat#HG10041-ACR) and the NF-κB reporter gene (pGL4.32[luc2P / NF-κB-RE / Hygro] Vector, purchased from Promega, Cat#E849A) to generate HEK293-Hu4-1BB / NF-κB double-transfected cells. 4-1BB activation can be characterized by the activation level of the NF-κB signaling pathway. HEK293 cells were transiently transfected with the FcγRIIb plasmid (CD32B / Fcgr2b cDNA ORF Clone, Human, N-His tag; purchased from Sino Biological, Cat#HG10259-NH) to obtain HEK293 cells that highly express FcγRIIb. The cell culture medium was DMEM (Gibco, Cat#11995065) containing 10% fetal bovine serum. HEK293-Hu4-1BB / NF-κB cells (2×10 6 / mL) were plated into 96-well cell culture plates at 50 μL, and 40 μL of culture medium or HEK293 cells expressing FcγRIIb (2.5×10 6 / mL), 10×10 μL of serially diluted anti-4-1BB antibody to be tested was added to each well and incubated at 37°C for 6 hours. The cells were removed and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat#G7940) was added to each well. The cells were incubated in the dark for 5 minutes. The fluorescence signal was measured using an Envision microplate reader (PerkinElmer, 2150) and the EC was calculated. 50 The EC values ​​and Emax values ​​(relative to the fluorescence intensity of the group without antibody) were used. 50 The in vitro cell agonist activity of the anti-4-1BB antibody was evaluated using the 4-1BB value. The results are shown in Figure 2 and Table 4.

[0323] The results showed that when FcγRIIb (ie, CD32b) positive cross-linking was not added, the anti-4-1BB antibodies involved showed that the activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway was much weaker than that of the Urelumab control, indicating that the anti-4-1BB antibodies disclosed herein are safer; after adding FcγRIIb cross-linking, the anti-4-1BB antibodies involved showed significant activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway, and C5 had the strongest activation ability and was equivalent to the Urelumab control. Based on the comprehensive activity results, the sequence C5 with low background activation before FcγRIIb cross-linking and stronger activation activity after FcγRIIb cross-linking was screened and humanized.

[0324] Table 4. EC of agonistic activity of anti-4-1BB antibodies on 4-1BB / NF-κB luciferase reporter gene 50 value (Note: "-" means the detection value exceeds the detection limit).

[0325] Example 3. Modification of anti-4-1BB antibodies

[0326] 1. Humanization

[0327] The C5 sequence's CDRs and FR regions (human framework) were numbered using the Kabat numbering system. The FR1, FR2, and FR3 sequences were then compared against an antibody germline database to obtain human germline FR templates with high homology. FR1 was derived from IGHV3-64*04, FR2 from IGHV3-23*03, and FR3 from IGHV3-74*01. These human germline FR regions were inserted into the original sequence to reduce immunogenicity in humans. Key amino acids that affect antibody structure and function were backmutated to restore binding and activity. The humanized sequences are shown below.

[0328] >C5_V1

[0329] >C5_V2

[0330] >C5_V3

[0331] (Note: The underlined area is the CDR region)

[0332] The above three humanized sequences were connected to human IgG1-Fc (SEQ ID NO: 14) respectively. Plasmids were constructed, transiently transfected, expressed and purified. The specific process is as follows: 60 μL of culture medium was diluted with transfection reagent and mixed with 15 μg of plasmid. After incubation at 37°C for 15 minutes, the mixed transfection solution was added dropwise to 30 mL of cell fluid, placed on a shaker for expression for one week, and the supernatant was collected. It was then subjected to Protein A affinity purification, eluted with citric acid buffer (pH 3.4), and finally dialyzed with 1xPBS buffer and frozen.

[0333] 2. Removal of TCE sites

[0334] We subsequently performed T-cell epitope (TCE) prediction on the C5_V2 sequence and modified the CDR3 sequence of C5_V2 based on the predicted results to reduce the number of TCEs. We mutated the F in the CDR3 region to Y (F99Y, according to the Kabat numbering system) to obtain a TCE-optimized version of the C5_V2 sequence, named C5_V2-YTI. Its sequence is as follows:

[0335] >C5_V2-YTI

[0336] That is, according to the Kabat numbering system, the amino acid sequence of CDR1 in C5_V2-YTI is shown in SEQ ID NO: 11, the amino acid sequence of CDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of CDR3 is shown in HPLTYTIATMNDYDY (SEQ ID NO: 22).

[0337] The above C5_V2-YTI sequence was linked to human IgG1-Fc (SEQ ID NO: 14), and a plasmid was constructed, transiently transfected, expressed, and purified.

[0338] 3. Amino acid modification of the VH framework (FR) of the anti-4-1BB single-domain antibody, the entire text of WO2023093899 was introduced to obtain C5_V2-YTI-AA (corresponding to C5_V2-YTI-53 shown in the 30th sequence in WO2023093899).

[0339] Example 4. Antigen Binding Activity and Agonist Activity Detection of Modified Anti-4-1BB Antibodies

[0340] 1. Detection of binding ability to 4-1BB antigen

[0341] The antigen binding activity of the humanized antibodies was detected using the FACS detection method described in Example 2. The results are shown in Figure 3 and Table 5.

[0342] The results showed that the humanized anti-4-1BB antibodies involved had different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, and C5_V1, C5_V2, and C5_V3 all maintained good binding activity.

[0343] Table 5. Affinity EC of anti-4-1BB antibodies before and after humanization and human 4-1BB high-expressing cell lines 50 value

[0344] 2. Detection of agonist activity on the 4-1BB signaling pathway

[0345] The humanized antibody was used to detect 4-1BB / NF-κB signaling activation in the NF-κB luciferase reporter gene assay as described in Example 2. The results are shown in Figure 4 and Table 6.

[0346] The results showed that after the addition of FcγRIIb cross-linking, the humanized anti-4-1BB antibodies involved showed activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway, and the activation ability of C5, C5_V1, C5_V2, and C5_V3 was comparable to that of the Urelumab control.

[0347] Table 6. Activation of NF-κB signaling pathway by humanized anti-4-1BB antibodies before and after administration (Note: Emax fold change <1.5 is defined as "-"; 1.5 to 2 is defined as "+"; 2 to 2.5 is defined as "++"; 2.5 to 3 is defined as "+++"; >3 is defined as "++++".)

[0348] 3. Detection of the binding ability of humanized 4-1BB antibody to antigen after removal of TCE

[0349] The humanized antibody C5_V2 was optimized for TCE removal, and the resulting antibody was C5_V2-YTI (see Example 3 for details). The antigen binding activity of C5_V2 and C5_V2-YTI was compared using the FACS detection method described in Example 2. The results are shown in Figure 5 and Table 7. The results showed that the C5_V2-YTI in question maintained good binding activity to 4-1BB on the surface of HEK293-Hu4-1BB cells, comparable to that of C5_V2.

[0350] Table 7. Affinity EC of C5_V2, C5_V2-YTI and human 4-1BB high-expressing cell lines 50 value

[0351] Example 5. Process for obtaining anti-CD16A nanobody

[0352] 1. Screening of the Natural Library of Human CD16A

[0353] Research has revealed that humans also have a gene called FcγRIIIb (CD16B) that is very similar in sequence to CD16A, with over 97% homology. CD16B is primarily expressed on neutrophils, with a small amount of soluble protein also present in the blood. Therefore, we need to screen for specific antibodies against CD16A, as these antibodies do not recognize CD16B. First, a camel natural library was subjected to a first round of panning using biotin-tagged human CD16A protein (CDA-H82E8, Biopsies) at a concentration of 5 μg / mL. Phages were eluted with Gly-HCl at pH 2.2 to obtain the first round of phages. The phages were then amplified and subjected to a second round of panning. In this second round, negative selection was performed using 5 μg / mL of biotin-tagged human CD16B protein (CDB-H82E4) to remove phages that bind to human CD16B. Phages were then screened again with 3 mg / mL of human CD16A protein, and the second round of panning products were eluted. Finally, the second round of panning products were subjected to phage ELISA, and phages that recognized human CD16A but not human CD16B were selected for sequencing to obtain the target antibody sequence.

[0354] 2. Camel Immunization and Screening of Human CD16A

[0355] Camel immunization combined with phage display was used to screen and prepare CD16A-specific antibodies. The immunization strategy is shown in Table 8.

[0356] Table 8. Immunization strategy options

[0357] PBMCs were isolated from the blood sample collected from the fourth immunization, and a library was constructed. The screening strategy was consistent with the screening of the natural camel library in Example 2.1. After screening, two candidate molecules, 34 and 501, were selected for subsequent development. Their sequences are as follows:

[0358] >34

[0359] >501

[0360] Table 9. CDRs of 34 and 501 (Kabat numbering rules)

[0361] 3. Cell Binding Validation of Candidate Molecules

[0362] The candidate molecules were tested for their binding ability to CHO cell lines overexpressing human CD16A-V176 (human CD16A-V176 protein sequence: AAH36723.1), CD16B (human CD16B protein sequence: O75015.2), or cynomolgus macaque CD16 (monkey CD16 sequence: NP_001270121.1). VHH-2, a positive antibody with high affinity for human CD16B, was selected as a control (from patent US20190276554). The candidate molecules showed sub-nM to nM binding to both human CD16A and monkey CD16, but very weak binding to human CD16B (Figure 6).

[0363] The above VHH-2 was linked to a human IgG1-Fc (mutations are underlined) with C220A, L234A, and L235A mutations (numbered according to the Eu system), and VHH-2 was placed at the N-terminus of Fc. The sequence was named VHH-2-LALA and is as follows:

[0364] >Human CD16B positive antibody VHH-2-LALA

[0365] >Human CD16A-V176 (AAH36723.1) amino acid sequence:

[0366] >Human CD16B (O75015.2)

[0367] >Cynomolgus macaque CD16 (NP_001270121.1)

[0368] 4. Humanization of candidate molecules

[0369] Anti-human CD16A antibodies 34 and 501 were humanized, and their binding activity was subsequently evaluated on a human CD16A-overexpressing cell line. The humanization template for 34 was IGHV3-23*04, upon which several back mutations were made. The humanization template for 501 was IGHV3-20*04, upon which several back mutations were made. The amino acid sequences of the humanized versions of 34 and 501 are as follows:

[0370] >34H1

[0371] >34H2

[0372] >34H3

[0373] >34H4

[0374] >34H5

[0375] >501V1

[0376] >501V2

[0377] >501V3

[0378] >501-V3NQ

[0379] Among them, CDR1 of 501-V3NQ is shown in SEQ ID NO: 28, CDR2 is shown in CINWQGGRTQYGDSVKG (SEQ ID NO: 44), and CDR3 is shown in SEQ ID NO: 30.

[0380] 5. Cell Binding Identification of Humanized Candidate Molecules

[0381] The above antibodies were linked to human IgG1-Fc and transiently expressed by transfection, followed by cell binding assay. 34H3 and 501-V3NQ were selected for subsequent development and bispecific antibody combinations. The cell binding results for 34H3 and 501-V3NQ are shown in Figures 7A and 7B, both of which were similar to those of the parental antibodies before humanization.

[0382] Example 6. Preparation of CLDN18.2 / 4-1BB multifunctional antibody

[0383] 1. Design, Expression, and Purification of a CLDN18.2 / 4-1BB Bispecific Antibody (CLDN18.2 x 4-1BB)

[0384] Based on the results of the humanized anti-4-1BB nanoantibody screening, the C5_V2 clone and the CLDN18.2 monoclonal antibody sequence (derived from WO2020200196A1) were selected to construct the CLDN18.2 x 4-1BB bispecific antibody. The sequence of CLDN18.2 monoclonal antibody 1903 is as follows:

[0385] >CLDN18.2 mAb 1903 heavy chain (SEQ ID NO: 45)

[0386] >CLDN18.2 mAb 1903 light chain (SEQ ID NO: 46)

[0387] Among them, the italicized part in the heavy chain is the heavy chain constant region, and the italicized part in the light chain is the light chain constant region.

[0388] The CDRs of 1903 defined according to the Kabat numbering convention are:

[0389] HCDR1 is SYWMH (SEQ ID NO: 57), HCDR2 is MIHPNSGSTNYNEKFKG (SEQ ID NO: 58), and HCDR3 is LKTGNSFDY (SEQ ID NO: 59);

[0390] LCDR1 is KSSQSLLNSGNQKNYLT (SEQ ID NO: 60), LCDR2 is WASTRES (SEQ ID NO: 61), and LCDR3 is QNAYTYPFT (SEQ ID NO: 62).

[0391] The antibody heavy and light chain variable regions and Fc segment sequences are as follows:

[0392] >1903 VH (SEQ ID NO: 63)

[0393] >1903 VL (SEQ ID NO: 64)

[0394] >IgG1 Fc (containing S239D, I332E mutations) (SEQ ID NO: 65)

[0395] The bispecific antibody molecule uses the aforementioned CLDN18.2 monoclonal antibody sequence as its backbone, with its Fc carrying the S239D / I332E mutations. A C5_V2 residue is fused to each of the two CLDN18.2 heavy chains at the C-terminus, using a (G4S)2 linker, resulting in the bispecific antibody molecule 1903 x C5_V2. Using 1903 x C5_V2 as an example, the naming convention for the molecules in this disclosure is as follows: 1903 represents the variable region of the CLDN18.2 monoclonal antibody, and C5_V2 represents the 4-1BB nanobody clone number. The bispecific antibody molecule 1903 x C5_V2-YTI was obtained by replacing C5_V2 with C5_V2-YTI after TCE removal. The antibody structure is shown in Figure 8. The 1903 x C5_V2-YTI bispecific antibody molecule was further optimized by altering two amino acid residues at the C-terminus of C5_V2-YTI to remove the pre-ADA residue. The optimized bispecific antibody molecule was named 1903 x C5_V2-YTI-AA.

[0396] The amino acid sequence of the aforementioned bispecific antibody molecule is shown below:

[0397] >1903 x C5_V2 heavy chain (SEQ ID NO: 47)

[0398] >1903 x C5_V2-YTI heavy chain (SEQ ID NO: 48)

[0399] >1903 x C5_V2-YTI-AA heavy chain (SEQ ID NO: 49)

[0400] The heavy chain constant region is in italics, and the linker is underlined. The light chain amino acid sequences of 1903 x C5_V2, 1903 x C5_V2-YTI, and 1903 x C5_V2-YTI-AA are all shown in SEQ ID NO: 46.

[0401] Transient transfection and expression: Taking a 30 mL expression system as an example, dilute the culture medium with 60 μL of transfection reagent and mix with 15 μg of plasmid. Incubate at 37°C for 15 minutes. Add the mixed transfection solution dropwise while shaking the cell solution. Incubate on a shaker for one week. Collect the supernatant and centrifuge at 8000 rpm for 5 minutes.

[0402] Antibody purification: First, equilibrate a Protein A affinity chromatography column with 1xPBS at a flow rate of 1 mL / min for 20 mL. Load the column at a flow rate of 1 mL / min. Wash the column with 1xPBS at a flow rate of 1 mL / min for 20 mL. Elute the column with citrate buffer (pH 3.4) at 1 mL / min, collect the protein, and read the absorbance at 280 nm using a NanoDrop analyzer. Finally, transfer the high-concentration protein to a dialysis bag and dialyze it in a beaker of 1xPBS. After testing, the target antibody molecule was obtained.

[0403] 2. Design, Expression, and Purification of a CLDN18.2 / 4-1BB / CD16A Trispecific Antibody (CLDN18.2x4-1BBxCD16A)

[0404] Based on the screening results of the humanized anti-4-1BB nanoantibody, the C5_V2 clone was selected, and based on the screening results of the humanized anti-CD16A nanoantibody, the 34H3 clone was selected to construct the CLDN18.2 x 4-1BB x CD16A trispecific antibody together with the aforementioned CLDN18.2 monoclonal antibody 1903.

[0405] The trispecific antibody molecule uses the CLDN18.2 monoclonal antibody 1903 as its backbone and is of the IgG1 subtype. Its Fc adopts the S239D / I332E mutations. A C5_V2 moiety is fused to the C-terminus of each of the CLDN18.2 heavy chains, using a (G4S)2 linker. A 34H3 moiety is fused to the C-terminus of each of the light chains, using a (G4S)3 linker. This construct creates the multispecific antibody molecule 1903 x C5_V2 x 34H3. Using 1903 x C5_V2 x 34H3 as an example, the molecule naming convention is 1903, indicating that the variable region of the CLDN18.2 monoclonal antibody 1903 is used. C5_V2 is the clone number of the 4-1BB nanobody, and 34H3 is the clone number of the CD16A nanobody. The trispecific antibody molecule 1903 x C5_V2-YTI x 34H3 was constructed by replacing C5_V2 with C5_V2-YTI after TCE removal. The antibody structure is shown in Figure 9. This 1903 x C5_V2-YTI x 34H3 trispecific antibody molecule was further optimized by modifying two amino acids at the C-terminus of C5_V2-YTI and 34H3 to remove the pre-ADA. The optimized trispecific antibody molecule was named 1903 x C5_V2-YTI x 34H3-AA.

[0406] The amino acid sequence of the aforementioned trispecific antibody molecule is shown below:

[0407] >1903 x C5_V2 x 34H3 heavy chain (SEQ ID NO: 50)

[0408] >1903 x C5_V2 x 34H3 light chain (SEQ ID NO: 51)

[0409] >1903 x C5_V2-YTI x 34H3 heavy chain (SEQ ID NO: 52)

[0410] >1903 x C5_V2-YTI x 34H3 light chain (SEQ ID NO: 51)

[0411] >1903 x C5_V2-YTI x 34H3-AA heavy chain (SEQ ID NO: 53)

[0412] >1903 x C5_V2-YTI x 34H3-AA light chain (SEQ ID NO: 54)

[0413] In the above sequences, the italicized portion in the heavy chain represents the heavy chain constant region, the italicized portion in the light chain represents the light chain constant region, and the underlined portion represents the linker.

[0414] The method of Part 1 of Example 6 was used for transient cell transfection and expression, and antibody purification to obtain the target antibody molecule.

[0415] Example 7. Antigen Binding Activity of C5_V2-YTI

[0416] We compared the antigen binding activity of C5_V2, 1903 x C5_V2-LALA, and 1903 x C5_V2-YTI-LALA against human 4-1BB. C5_V2 was linked to a human IgG1-Fc with C220A / S267E / L328F mutations, the sequence of which is shown in SEQ ID NO: 17. 1903 x C5_V2-LALA was obtained by introducing the L234A / L235A mutations into the Fc of the 1903 x C5_V2 heavy chain (SEQ ID NO: 47), without the S239D / I332E mutations (SEQ ID NO: 55). The light chain remained as shown in SEQ ID NO: 46. 1903 x C5_V2-YTI-LALA is derived from the 1903 x C5_V2-YTI heavy chain (SEQ ID NO: 48) by introducing the L234A / L235A mutations into the Fc region. The Fc region lacks the S239D / I332E mutations (SEQ ID NO: 56), while the light chain remains as shown in SEQ ID NO: 46. The sequence is as follows:

[0417] >1903 x C5_V2-LALA heavy chain (SEQ ID NO: 55)

[0418] >1903 x C5_V2-YTI-LALA heavy chain (SEQ ID NO: 56)

[0419] The heavy chain sequences of 1903 x C5_V2-LALA and 1903 x C5_V2-YTI-LALA are both shown in SEQ ID NO: 46.

[0420] The binding activity of the modified antibody of C5_V2-YTI to human 4-1BB protein was detected by FACS experiment. HEK293-Hu4-1BB cells were obtained by overexpressing human 4-1BB protein in HEK293 cells. The cell culture medium was DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100μg / mL hygromycin B. After recovery, the cells were passaged to adjust the state. The experimental culture medium was sterile PBS (phosphate buffer, pH7.40) containing 2% fetal bovine serum. The HEK293-Hu4-1BB cells were washed twice with experimental culture medium, and 1×10 cells were added to each well. 5 Cells were seeded in 96-well plates and tested at varying concentrations. After incubation at 4°C for 1 hour, the cells were washed twice with experimental medium. Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat# 209-605-098) was then added. After two washes, the fluorescence signal was measured by flow cytometry. The results are shown in Figure 10 and Table 10.

[0421] The FACS test results showed that the 1903 x C5_V2-YTI-LALA antibody involved had a strong binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, which was comparable to 1903 x C5_V2-LALA and C5V2, indicating that C5_V2 and C5_V2-YTI had the same binding ability to 4-1BB.

[0422] Table 10. Results of C5_V2-YTI modified antibodies binding to HEK293-Hu4-1BB cells (Note: “-” means it exceeds the detection limit)

[0423] Example 8. 4-1BB / NF-κB luciferase reporter gene detection experiment of C5_V2-YTI

[0424] The agonist activity of the antibody was assessed using the 4-1BB / NF-κB reporter gene. HEK293 cells (ATCC CRL-1573) were transiently transfected with the human 4-1BB gene (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; Sino Biological, Cat#HG10041-ACR) and the NF-κB genome (pGL4.32[luc2P / NF-κB-RE / Hygro] Vector, Promega, Cat#E849A) to generate HEK293-Hu4-1BB / NF-κB double-transfected cells. Activation of Hu4-1BB can be characterized by measuring the level of activation of the NF-κB signaling pathway. NUGC4-hi18.2 cells were obtained by stably transfecting NUGC4 cells to express human CLDN18.2 protein. The cell culture medium was RPMI1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum and 10 μg / mL puromycin. HEK293-Hu4-1BB / NF-κB cells (2×10 6 / mL) were plated into 96-well cell culture plates at 50 μL, and 40 μL of culture medium or NUGC4 cells expressing CLDN18.2 (2.5×10 6 / mL), add 10×10 μL of serially diluted test antibody to each well and incubate at 37°C for 6 hours. Remove the cells, add an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat#G7940) to each well, incubate in the dark for 5 minutes, measure the fluorescence signal with an Envision microplate reader, and calculate the EC 50 The values ​​and Emax values ​​(relative to the fluorescence intensity of the no antibody group) were measured, and the in vitro cell agonist activity of the anti-4-1BB antibody was evaluated. The results are shown in Figure 11 and Table 11.

[0425] The results showed that the involved 1903 x C5_V2-YTI-LALA antibody had no background activation, but its 4-1BB agonist activity after cross-linking through CLDN18.2 was comparable to that of 1903 x C5_V2-LALA, indicating that C5_V2 and C5_V2-YTI have the same activation ability for 4-1BB.

[0426] Table 11. CLDN18.2-dependent 4-1BB / NF-κB luciferase reporter gene assay results of the C5_V2-YTI modified antibody

[0427] Example 9. Detection of antigen binding affinity of CLDN18.2 / 4-1BB multifunctional antibody

[0428] Surface plasmon resonance (SPR) was used to measure the affinity of the CLDN18.2 / 4-1BB multifunctional antibody for antigens. A CM5 sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Anti-human IgG (Fc) antibody was prepared at a concentration of 30 μg / mL in 10 mM sodium acetate buffer (pH 5.0). The Immobilization program was selected for automated amino-coupling of the anti-human IgG (Fc) antibody onto the chip channel. Each test antibody was prepared in HBS-EP+ buffer as a ligand and captured by the anti-human IgG (Fc) antibody on the chip channel. Human 4-1BB protein (Acro Biosystems, 41B-H522a) and cynomolgus monkey 4-1BB protein (Acro Biosystems, 41B-C52H4) were used as antigens (i.e., analytes). They were prepared with HBS-EP+ buffer solution, and the analytes were diluted 2-fold. The diluted antibodies were passed through the experimental channel and the reference channel at a flow rate of 30 μL / min, bound for 1 minute, and dissociated for 15 minutes. 10mM Glycine pH1.5 (GE Healthcare, BR-1003-54) was selected and run for 30 seconds at a flow rate of 10 μL / min of regeneration buffer. The instrument used was Zeba Spin Desalting Columns (Thermo, 89882), and the data were analyzed using Biacore 8K evaluation software.

[0429] The results are shown in Table 12, which show that the antigen binding affinity of the CLDN18.2 x 4-1BB multifunctional antibody involved in the study for human and cynomolgus monkey 4-1BB proteins is comparable to that of the parental 4-1BB VHH antibody C5_V2.

[0430] Table 12. Biacore affinity of CLDN18.2 / 4-1BB multifunctional antibodies for 4-1BB (1:1 binding)

[0431] Example 10. Antigen Binding Activity Detection of CLDN18.2 / 4-1BB Multifunctional Antibody

[0432] FACS assays were used to detect the binding activity of the CLDN18.2 / 4-1BB multifunctional antibody to human CLDN18.2 / CD16A / 4-1BB proteins. NUGC4-hi18.2 cells were obtained by stably transfecting NUGC4 cells to express human CLDN18.2 protein. The cell culture medium was RPMI1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum and 10 μg / mL puromycin. CHO-K1-CD16A V176 cells were obtained by overexpressing human CD16A V176 protein in CHO-K1 cells. The cell culture medium was Ham's F12 + GlutaMAX. TM -I (Gibco, Cat#31765035), containing 10% fetal bovine serum and 200μg / mL hygromycin B. HEK293-Hu4-1BB cells were obtained by overexpressing human 4-1BB protein in HEK293 cells. The cell culture medium was DMEM (Gibco, Cat#11965092), containing 10% fetal bovine serum and 100μg / mL hygromycin B. After recovery, the cells were passaged to adjust the state. The experimental culture medium was sterile PBS (phosphate buffer, pH7.40) containing 2% fetal bovine serum. NUGC4-hi18.2, CHO-K1-CD16A V158 or HEK293-Hu4-1BB cells were washed twice with experimental culture medium, and 1×10 cells were added to each well. 5 Cells were seeded in 96-well plates and tested at varying concentrations. After incubation at 4°C for 1 hour, the cells were washed twice with experimental medium. Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat# 209-605-098) was then added. After two washes, fluorescence signals were measured by flow cytometry. The results are shown in Figures 12A to 12C and Tables 13 to 15.

[0433] FACS analysis showed that the CLDN18.2 / 4-1BB multifunctional antibody had strong binding to CLDN18.2 on the surface of NUGC44-hi18.2 cells, comparable to the binding of CLDN18.2 monoclonal antibody 1903 and the control antibody TJ-CD4B, and stronger than the control antibody IMAB362. The CLDN18.2 / 4-1BB multifunctional antibody had good binding to CD16A V176 on the surface of CHO-K1-CD16A V176 cells, stronger than the control antibody IMAB362. The CLDN18.2 / 4-1BB multifunctional antibody had strong binding to 4-1BB on the surface of HEK293-Hu4-1BB cells, comparable to the binding of 4-1BB monoclonal antibody C5_V2 and slightly stronger than TJ-CD4B.

[0434] Table 13. Results of CLDN18.2 / 4-1BB multifunctional antibodies binding to NUGC4-hi18.2 cells

[0435] Table 14. Results of CLDN18.2 / 4-1BB multifunctional antibody binding to CHO-K1-CD16A V176 cells

[0436] Table 15. Results of CLDN18.2 / 4-1BB multifunctional antibodies binding to HEK293-Hu4-1BB cells (Note: “-” means it exceeds the detection limit)

[0437] Example 11. Experimental study on the induction of antibody-mediated cell killing (ADCC) by CLDN18.2 / 4-1BB multifunctional antibody in vitro

[0438] Antibody-mediated ADCC activity of NK cells against target cells expressing CLDN18.2 was assessed using a lactate dehydrogenase (LDH) assay. NUGC4-hi18.2 cells were derived from NUGC4 cells stably transfected with human CLDN18.2 protein and cultured in RPMI1640 (Gibco, Cat#10491A-01) supplemented with 10% inactivated fetal bovine serum and 10 μg / mL puromycin. SNU601 cells were purchased from Nanjing Kebai Biotechnology (CBP60507) and cultured in RPMI1640 (Gibco, Cat#10491A-01) supplemented with 10% fetal bovine serum. Cryopreserved PBMCs were isolated from fresh human blood and thawed and cultured in RPMI1640 (Gibco, Cat#10491A-01) supplemented with 10% fetal bovine serum at 37°C overnight. The next day, different target cells were digested and resuspended in phenol red-free RPMI1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum, and the density was adjusted to 2×10 5 cells / mL; 50 μL / well was then seeded into a 96-well plate, and 10×10 μL of serially diluted test antibody was added. The cells were incubated in a 37°C, 5% CO2 incubator for 0.5 h.

[0439] PBMCs were collected and resuspended in phenol red-free RPMI1640 containing 2% fetal bovine serum. Depending on the target cell, an appropriate effector-to-target ratio was used and the cell density was adjusted. 40 μL / well was seeded into the above-mentioned experimental plate and incubated in a 37°C, 5% CO2 incubator for 4 hours. The cell culture plate was removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant. The cell culture supernatant was then analyzed using CytoTox. LDH levels were measured using a Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). For detailed procedures, refer to the kit instructions.

[0440] The results are shown in Figures 13A to 13D and Table 16, which show that the CLDN18.2 / 4-1BB multifunctional antibodies involved have good ADCC activity against target cells with different CLDN18.2 expression levels, which is stronger than the control antibody IMAB362. Among them, SC190061 (176F) is the PBMC donor number of the CD16A-F176 variant, and S2001102 (176V) is the PBMC donor number of the CD16A-V176 variant.

[0441] Table 16. Results of CLDN18.2 / 4-1BB multifunctional antibody-induced NK cell killing of target cells expressing CLDN18.2 (Note: “-” means it exceeds the detection limit)

[0442] Example 12. Experimental study on the induction of antibody-mediated phagocytosis (ADCP) by CLDN18.2 / 4-1BB multifunctional antibody in vitro

[0443] PBMCs were isolated from fresh human blood and then sorted for CD14 using human CD14 microbeads (Miltenyi Biotec, 130-050-201). + Monocytes. These CD14 + Monocytes were cultured in RPMI1640 (Gibco, Cat#10491A-01), a macrophage differentiation medium, containing 50 ng / mL recombinant human macrophage colony-stimulating factor (rhM-CSF, PeproTech, Cat#300-25) and 10% fetal bovine serum. After 6 days of differentiation, the macrophages became adherent and developed tentacles. The macrophages were trypsinized for 5 minutes, gently scraped with a spatula, and resuspended in RPMI1640 containing 10% fetal bovine serum at a density of 4 × 10 5cells / mL. They were then inoculated in 96-well plates and incubated overnight at 37°C, 100 μL / well. The next day, NUGC4-hi18.2 cells were labeled with CellTrace Far Red (Invitrogen, C34564) at 37°C for 15 minutes. After washing twice with PBS, NUGC4-hi18.2 was added to the wells inoculated with macrophages at a ratio of 50 μL / well and 5 NUGC4-hi18.2 per macrophage. 4×50 μL of gradiently diluted antibodies to be tested were added to phagocytose the target cells for 4 hours. After the phagocytosis was completed, the cells were washed three times with PBS, and then FITC anti-human / mouse CD11b (Tonbo, 35-0112-M100) was added in a certain proportion for staining for 30 minutes. After washing twice with PBS, flow cytometry was used for analysis. Far Red was evaluated after gating on CD11b-positive cells. + / CD11b + Phagocytosis was measured as the percentage of double-positive cells.

[0444] The results are shown in Figure 14 and Table 17, which show that the CLDN18.2 / 4-1BB multifunctional antibodies involved have good ADCP function, which is stronger than the control antibody IMAB362.

[0445] Table 17. Results of CLDN18.2 / 4-1BB multifunctional antibody-induced macrophage phagocytosis of tumor cells (Note: “-” means it exceeds the detection limit)

[0446] Example 13. CLDN18.2-dependent 4-1BB / NF-κB luciferase reporter gene assay using the CLDN18.2 / 4-1BB multifunctional antibody

[0447] The NF-κB luciferase reporter gene assay described in Example 8 was used to detect CLDN18.2-dependent 4-1BB / NF-κB signaling activation.

[0448] The results are shown in Figure 15 and Table 18, which show that the CLDN18.2 / 4-1BB multifunctional antibodies involved have no background activation, and the agonist activity of CLDN18.2 dependent on the 4-1BB / NF-κB signaling pathway is comparable to that of TJ-CD4B.

[0449] Table 18. Results of CLDN18.2-dependent 4-1BB / NF-κB luciferase reporter gene assay using CLDN18.2 / 4-1BB multifunctional antibodies (Note: “-” means it exceeds the detection limit)

[0450] Example 14. T lymphocyte activation experiment

[0451] Freshly isolated and purified PBMCs were resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum and the density was adjusted to 1×10 6 NUGC4-hi18.2 cells were obtained by stably transfecting NUGC4 cells to express human CLDN18.2 protein. The cell culture medium was RPMI1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum and 10 μg / mL puromycin. After recovery, the cells were passaged to adjust the cell state and the density was adjusted to 1.25×10 6 96-well plates were coated with 0.25 μg / mL anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85), 100 μL per well, incubated at 37°C for 2 hours, and then washed with PBS to remove residual antibodies. Subsequently, 100 μL of PBMC cells (1×10 5 cells / well) and 80 μL of culture medium or NUGC4-hi18.2 cells (1×10 5 Cells (cells / well) were seeded into OKT3-coated 96-well plates. Test samples at 10x concentrations were added at 20 μL / well and incubated at 37°C, 5% CO2 for 3 days. The plates were removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant. IL-2 levels were measured using a human IL-2 detection kit (Cisbio, Cat# 62HIL02PEG). For detailed procedures, refer to the reagent instructions.

[0452] The results are shown in Figures 16A and 16B, which show that in both PBMC donors, after CLDN18.2 cross-linking, the CLDN18.2 / 4-1BB multifunctional antibodies involved can activate IL-2 secretion, which is comparable to the control antibody TJ-CD4B.

[0453] Example 15. Experiment on NK cell killing of T cells

[0454] PBMCs were isolated from fresh human blood and then analyzed using EasySep TM T cells were isolated using the Human T Cell Enrichment Kit (Stemcell, Cat#17951). T75 culture flasks (Corning, Cat#430641) were coated with 0.25 μg / mL anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85) and incubated at 37°C for 2 hours. Residual antibody was then washed with PBS to remove the remaining T cells. Freshly isolated T cells were resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum and seeded onto OKT3-coated T75 culture flasks at a density of 2 × 106 After 48 hours of incubation, activated T cells were labeled with CellTrace Far Red (Invitrogen, C34564) at 37°C for 15 minutes, washed twice with PBS, and resuspended in phenol red-free RPMI1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum at a density of 2 × 10 5 cells / mL; 50 μL / well was then seeded into a 96-well plate, and 10×10 μL of serially diluted test antibody was added. The cells were incubated in a 37°C, 5% CO2 incubator for 0.5 h.

[0455] Freshly recovered PBMCs from the same donor were collected and resuspended in phenol red-free RPMI1640 containing 2% fetal bovine serum. The cell density was adjusted according to the effector-target ratio of PBMC: activated T cells = 25:1. Fresh PBMCs were inoculated into the above experimental plates at 40 μL / well and incubated in a 37°C, 5% CO2 incubator for 6 hours. After incubation, the cell culture plates were removed and PI (Aibixin, abs9358) was added according to a certain ratio for staining for 10 minutes. After washing twice with PBS, flow cytometry analysis was performed. PI was evaluated after CellTrace Far Red positive gating. + / Far Red + The cytotoxic effect of NK cells on activated T cells in PBMCs was measured by the percentage of double-positive cells.

[0456] The results are shown in Figure 17, which show that the CLDN18.2 x 4-1BB bispecific antibody 1903 x C5_V2-YTI involved did not induce NK cell killing of T cells, which was comparable to the isotype control IgG1; the CLDN18.2 x 4-1BB x CD16A multispecific antibody 1903 x C5_V2-YTI x 34H3 involved only showed slight induction of NK cell killing of T cells.

[0457] Example 16. Pharmacokinetics, safety and efficacy evaluation

[0458] 1. In vivo efficacy study of the CLDN18.2 / 4-1BB multifunctional antibody in the mouse colon cancer model MC38-hCLDN18.2

[0459] MC38-hCLDN18.2 cells (provided by Biocytogen) were cultured at 2×10 5 100 μL / mouse was inoculated subcutaneously into B-h4-1BB humanized mice (provided by Biocytogen) and the tumor was grown to approximately 102 mm. 3Twenty-four mice were randomly divided into four groups (six mice per group) based on tumor volume: vehicle, 1903 x C5_V2-YTI (0.26 mg / kg), 1903 x C5_V2-YTI x 34H3 (0.3 mg / kg), and TJ-CD4B (0.3 mg / kg). Dosing was performed every other day. Tumor volume was measured twice weekly during the dosing and observation period, and the measured values ​​were recorded.

[0460] Calculate tumor volume (TV) using the formula TV = 1 / 2 × a × b 2 , where a and b represent the long and short diameters of the measured tumor, respectively.

[0461] Relative tumor growth rate T / C% = (T-T0) / (C-C0) × 100%; tumor inhibition rate TGI% = 1-T / C%.

[0462] CR% (complete tumor regression ratio) = complete tumor regression (<102 mm 3 ) of mice / number of mice enrolled.

[0463] The results are shown in Figure 18A and Table 19, which show that the CLDN18.2 / 4-1BB multifunctional antibody has good anti-tumor activity and is better than the control antibody TJ-CD4B.

[0464] Table 19. Antitumor effects of different CLDN18.2 / 4-1BB multifunctional antibodies on mouse transplanted tumors

[0465] 2. Toxicity testing of the CLDN18.2 / 4-1BB multifunctional antibody in the mouse colon cancer model MC38-hCLDN18.2

[0466] CLDN18.2 / 4-1BB multifunctional antibody cells (provided by Biocytogen) were cultured at a concentration of 2 × 10 5 100 μL / mouse was inoculated subcutaneously into B-h4-1BB humanized mice (provided by Biocytogen) and the tumor was grown to approximately 102 mm. 3 Twenty-four mice were randomly divided into four groups (six mice per group) based on tumor volume: vehicle, 1903 x C5_V2-YTI (0.26 mg / kg), 1903 x C5_V2-YTI x 34H3 (0.3 mg / kg), and TJ-CD4B (0.3 mg / kg). Dosing was performed every other day. Body weights were measured twice weekly during dosing and observation, and the values ​​were recorded. Blood samples were collected on day 17 for AST and ALT assays.

[0467] The results are shown in Figures 18B and 19. The results showed that the body weight of the mice remained stable during the administration process, indicating that the CLDN18.2 / 4-1BB multifunctional antibody had no significant toxic side effects. ALT / AST assay results on day 17 showed that the CLDN18.2 / 4-1BB multifunctional antibody showed no significant hepatotoxicity at the effective anti-tumor dose.

[0468] 3. Single-dose pharmacokinetic study of the CLDN18.2 / 4-1BB multifunctional antibody in the mouse colon cancer model MC38-hCLDN18.2

[0469] MC38-hCLDN18.2 cells were cultured at 5×10 5 100 μL / mouse was inoculated subcutaneously into B-h4-1BB humanized mice (provided by Biocytogen) and the tumor was grown to approximately 110 mm. 3 Six mice were randomly divided into two groups according to tumor volume, with three mice in each group, namely 1903 (10 mg / kg) and 1903 x C5_V2 (12 mg / kg). Single dose was intravenous injection.

[0470] Blood was collected from the experimental mice at the following time points: 0.25h, 6h, 24h, 72h, 144h, 240h, 336h, 408h, and 504h after administration. Blood drug concentrations were detected by ELISA. The bottom of the plate was coated with goat anti-human IgG, Fc (Rockland, 609-101-017) and incubated at 4°C for 14-18 hours. After washing three times with 300μL PBS, a blocking reagent containing 3% BSA was added and incubated at room temperature for 1 hour. After washing three times with 300μL PBS, the serum to be tested was added and incubated at room temperature for 2 hours. After washing three times with 300μL PBS, 50μL goat anti-human IgG-HRP (BETHYL, A80-304P) solution was added to each well, the plate was sealed with a sealing film, and incubated at room temperature for 1 hour. The secondary antibody detection concentration was 1:20,000, and the dilution solution was 1% BSA-PBS + 0.5% mouse serum.

[0471] The results are shown in Figure 20 and Table 20. The CLDN18.2 / 4-1BB multifunctional antibodies exhibited pharmacokinetic parameters similar to those of monoclonal antibodies, primarily due to antibody distribution mediated by the CLDN18.2 terminus. The PK profile was similar to that of the CLDN18.2 parental monoclonal antibody 1903 at equimolar doses. These results indicate that the plasma concentrations of the CLDN18.2 / 4-1BB multifunctional antibodies in mice were not affected by the 4-1BB terminus, exhibiting typical pharmacokinetic characteristics of CLDN18.2-targeted antibodies.

[0472] Table 20. Pharmacokinetic parameters of CLDN18.2 / 4-1BB multifunctional antibody in h4-1BB transgenic mice (mean ± standard deviation)

Claims

1. A 4-1BB binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2, and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 10 and 18-21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown as SEQ ID NOs: 11, 12, 13, or as SEQ ID NOs: 11, 12, 22, respectively.

2. The 4-1BB binding protein according to claim 1, wherein the immunoglobulin single variable domain is humanized, affinity matured, T cell epitopes removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the heavy chain framework region of the human germline template used in the humanization process is IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01.

3. The 4-1BB binding protein according to claim 1 or 2, wherein the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 10, 18-21, or has at least 80% or at least 90% sequence identity thereto; Preferably, the 4-1BB binding protein is an anti-4-1BB nanobody or VHH.

4. The 4-1BB binding protein according to any one of claims 1 to 3, further comprising an immunoglobulin Fc region; Preferably, the Fc region is the Fc region of human IgG1, human IgG2 or human IgG4.

5. The 4-1BB binding protein according to any one of claims 1 to 4, comprising an amino acid sequence as shown in SEQ ID NO: 21 or having at least 80% or at least 90% identity thereto.

6. A CLDN18.2 / 4-1BB binding protein comprising: A first antigen binding domain that specifically binds 4-1BB; and a second antigen-binding domain that specifically binds to CLDN18.2, in, The first antigen binding domain comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2, and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 10 and 18-21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown as SEQ ID NOs: 11, 12, 13, or as SEQ ID NOs: 11, 12, 22, respectively.

7. The CLDN18.2 / 4-1BB binding protein according to claim 6, wherein the immunoglobulin single variable domain is humanized, affinity matured, T cell epitopes removed, antibody deamidation reduced, and / or antibody isomerization reduced; Preferably, the heavy chain framework region of the human germline template used in the humanization process is IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01.

8. The CLDN18.2 / 4-1BB binding protein according to claim 6 or 7, wherein the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 10, 18-21, or has at least 80% or at least 90% sequence identity thereto.

9. The CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 8, wherein The second antigen binding domain comprises: a heavy chain variable region (VH) and a light chain variable region (VL), The VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 63, The VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 64, The HCDRs and LCDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems; Preferably, the VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 57, 58, and 59, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 60, 61, and 62.

10. The CLDN18.2 / 4-1BB binding protein according to claim 9, wherein The VH comprises an amino acid sequence as shown in SEQ ID NO: 63 or a sequence having at least 80% or at least 90% identity thereto, The VL comprises an amino acid sequence as shown in SEQ ID NO: 64 or an amino acid sequence at least 80% or at least 90% identical thereto.

11. A CLDN18.2 / 4-1BB binding protein comprising: A first antigen binding domain that specifically binds 4-1BB; and a second antigen-binding domain that specifically binds to CLDN18.2, When the second antigen-binding domain does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB does not activate 4-1BB signaling; and the CLDN18.2 / 4-1BB binding protein has enhanced effector function; Preferably, the CLDN18.2 / 4-1BB binding protein comprises an Fc region, and the Fc region is used to enhance the effector function of the CLDN18.2 / 4-1BB binding protein; and / or, the CLDN18.2 / 4-1BB binding protein comprises a third antigen binding domain that specifically binds to CD16A; Preferably, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC); Preferably, the first antigen-binding domain and the second antigen-binding domain are the first antigen-binding domain and the second antigen-binding domain defined in any one of claims 6 to 10.

12. A CLDN18.2 / 4-1BB binding protein comprising: The first antigen-binding domain that specifically binds to 4-1BB, a second antigen binding domain that specifically binds to CLDN18.2; and A third antigen-binding domain that specifically binds to CD16A; Preferably, when the second antigen-binding domain that specifically binds to CLDN18.2 does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB cannot activate 4-1BB signaling; Preferably, the first antigen-binding domain and the second antigen-binding domain are the first antigen-binding domain and the second antigen-binding domain defined in any one of claims 6 to 10.

13. The CLDN18.2 / 4-1BB binding protein according to claim 12, wherein The third antigen binding domain comprises an immunoglobulin single variable domain comprising: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 23, 24, 35-43, The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown as SEQ ID NOs: 25, 26, 27, or as shown in SEQ ID NOs: 28, 29, 30, or as shown in SEQ ID NOs: 28, 44, 30, respectively.

14. The CLDN18.2 / 4-1BB binding protein according to claim 13, wherein The immunoglobulin single variable domain in the third antigen-binding domain is humanized, affinity matured, T cell epitopes removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the humanized process uses a human germline template whose heavy chain framework region is derived from IGHV3-23*04 or IGHV3-20*04.

15. The CLDN18.2 / 4-1BB binding protein according to any one of claims 12 to 14, wherein the amino acid sequence of the immunoglobulin single variable domain in the third antigen-binding domain is as shown in any one of SEQ ID NOs: 23, 24, 35-43, or has at least 80% or at least 90% sequence identity thereto.

16. The CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 15, further comprising an Fc region of an immunoglobulin; Preferably, the Fc region is the Fc region of human IgG1, human IgG2 or human IgG4; More preferably, the Fc region is an effector-enhanced Fc region.

17. The CLDN18.2 / 4-1BB binding protein according to claim 16, when the Fc region is the Fc region of human IgG1, the Fc region comprises any one or any combination of the following amino acid mutations: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239 D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 280H / 290S / 298V; 243L / 292P / 300L; 243L / 292P / 300L / 396L; 243L / 292P / 300L / 305I / 396L; 236A / 239D / 332E; 326A / 333A; 326W / 333S; 290E / 298G / 299A; 290N / 298G / 299A; 290E / 298G / 299A / 326E; or 290N / 298G / 299A / 326E; said mutations are defined according to the EU numbering system; Preferably, the Fc region contains any one or any combination of amino acid mutations selected from the group consisting of: S239D; S239E; S239K, F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D2 80H / K290S / S298D;D280H / K290S / S298V;F243L / R292P / Y300L;F243L / R292P / Y300L / P396L;F243L / R292P / Y300L / V305I / P396L;G236A / S239D / I332E;K326A / E333A;K326W / E333S;K290E / S298G / T299A;K290N / S298G / T299A;K290E / S298G / T299A / K326E;or K290N / S298G / T299A / K326E.

18. The CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 17, further comprising a linker; Preferably, the amino acid sequence of the linker is as follows (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h As shown, m and n are each independently selected from an integer of 1-8, and h is independently selected from an integer of 1-20; More preferably, the linker is a linker represented by (G4S)2 or (G4S)3.

19. The CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 18, wherein the second antigen-binding domain comprises a heavy chain and a light chain, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 45 or has at least 80% or at least 90% sequence identity thereto, The amino acid sequence of the light chain is shown in SEQ ID NO: 46 or has at least 80% or at least 90% sequence identity thereto.

20. The CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 19, comprising a first polypeptide chain and a second polypeptide chain selected from the following: (1) the amino acid sequence of the first polypeptide chain is as shown in any one of SEQ ID NOs: 47-49 or has at least 80% or at least 90% sequence identity thereto, The second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 46 or has at least 80%, at least 90% sequence identity thereto; (2) the amino acid sequence of the first polypeptide chain is as shown in any one of SEQ ID NOs: 50, 52, and 53, or has at least 80% or at least 90% sequence identity thereto, The amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 51 or 54, or has at least 80% or at least 90% sequence identity thereto.

21. A CD16A binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 23, 24, 35-43, The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown as SEQ ID NOs: 25, 26, 27, or as SEQ ID NOs: 28, 29, 30, or as SEQ ID NOs: 28, 44, 30, respectively.

22. The CD16A binding protein according to claim 21, wherein the immunoglobulin single variable domain is humanized, affinity matured, T cell epitopes removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the humanized process uses a human germline template whose heavy chain framework region is derived from IGHV3-23*04 or IGHV3-20*04.

23. The CD16A binding protein according to claim 21 or 22, wherein the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 23, 24, 35-43, or has at least 80% or at least 90% sequence identity thereto; Preferably, the CD16A binding protein is an anti-CD16A nanobody or VHH.

24. The CD16A binding protein according to any one of claims 21 to 23, further comprising a human immunoglobulin Fc region; Preferably, the Fc region is the Fc region of human IgG1, human IgG2 or human IgG4.

25. The CD16A binding protein according to any one of claims 21 to 24, which does not specifically bind to CD16B.

26. A polynucleotide encoding the CLDN18.2 / 4-1BB binding protein of any one of claims 6 to 20, the 4-1BB binding protein of any one of claims 1 to 5, or the CD16A binding protein of any one of claims 21 to 25; Preferably, the polynucleotide is DNA or RNA.

27. A vector comprising the polynucleotide of claim 26.

28. A host cell containing or expressing the polynucleotide of claim 26 or the vector of claim 27.

29. A method for preparing a CLDN18.2 / 4-1BB binding protein, a 4-1BB binding protein, or a CD16A binding protein, comprising: Expressing the polynucleotide of claim 26 or the vector of claim 27 in the host cell of claim 28, and isolating the expressed CLDN18.2 / 4-1BB binding protein, 4-1BB binding protein, or CD16A binding protein from the host cell; Optionally, further comprising purifying the CLDN18.2 / 4-1BB binding protein, 4-1BB binding protein, or CD16A binding protein step.

30. A pharmaceutical composition comprising the CLDN18.2 / 4-1BB binding protein of any one of claims 6 to 20, the 4-1BB binding protein of any one of claims 1 to 5, the CD16A binding protein of any one of claims 21 to 25, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

31. A method for treating cancer, comprising the steps of (1) or (2): (1) administering a therapeutically effective amount of the CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 20, or the pharmaceutical composition according to claim 30, to a subject in need thereof; preferably, the cancer is CLDN18.2-positive; or (2) administering a therapeutically effective amount of the 4-1BB binding protein of any one of claims 1 to 5, the CD16A binding protein of any one of claims 21 to 25, the polynucleotide of claim 26, the vector of claim 27, or the pharmaceutical composition of claim 30 to a subject in need thereof.

32. The method of claim 31, wherein the cancer is selected from the group consisting of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematological cancer, or any combination thereof.

33. The use as shown in (1) or (2) below: (1) Use of the CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 20, the polynucleotide according to claim 26, and the vector according to claim 27 for preparing a drug for treating cancer; preferably, the cancer is CLDN18.2-positive; or (2) Use of the 4-1BB binding protein according to any one of claims 1 to 5 or the CD16A binding protein according to any one of claims 21 to 25, the polynucleotide according to claim 26, or the vector according to claim 27 for preparing a drug for treating cancer.

34. The use according to claim 33, wherein the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematological cancer or any combination thereof.

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